FIELD: chemistry; biochemistry.
SUBSTANCE: invented here are proteins of the meningococcus bacteria Neisseria meningitides (mainly strain B), with immunogenic properties. The proteins have defined amino acid sequences, presented in the description, and are coded with corresponding nucleotide sequences. Description is also given of an antibody, specific to the indicated meningococcus proteins. These proteins, coding their nucleotide sequences, as well as the specific antibody, can be used as an active ingredient in compositions for treating or preventing infection caused by Neisseria meningitides. The presented proteins can be used as antigens for making effective vaccines, immunogenic compositions.
EFFECT: obtaining proteins, used as ingredients for making effective vaccines, immunogenic compositions.
11 cl, 2 tbl, 104 ex
The present invention relates to antigens of bacteria of the genus Neisseria.
Prerequisites for inventions
Bacteria Neisseria meningitidis and Neisseria gonorrhoeae are fixed by gram-negative bacteria-diplococci showing pathogenicity for humans. N.meningitidis form colonies in pharyngeal Department and cause meningitis (and, in some cases, septicaemia without meningitis); N.gonorrhoeae form colonies in the genital tract, causing gonorrhea. Despite the fact that they form colonies in different parts of the body and cause very different diseases, these two pathogen are very close to each other, although there is a distinct difference between the meningococcus and gonococcus associated with the presence of a polysaccharide capsule, which is present in all pathogenic meningococci.
Gonococcus N.gonorrhoeae causes approximately 800 thousand diseases per year for the period 1983-90, only in the United States (Chapter Meitzner & Cohen, 1997, "Vaccines Against Gonococcal Infection", In "New Generation Vaccines", 2d ed., ed. Levine, Woodrow, Kaper & Gobon, Marcel Dekker, NY, pp. 817-842). This disease is widespread, although death rates are low. Highly desirable is a vaccine against the pathogen gonorrhea, however, many such attempts have been unsuccessful. The main antigens candidates to create such vaccines are located on the surface of proteins, such as drinking,porins, assosiated with opacity proteins (Opas) and other surface proteins, such as Lip, Laz, IgA1-protease and transferrin-binding proteins. As the vaccine was offered to use the lipopolysaccharide (LOS) (Meitzner & Cohen, CIT. above).
Meningococcal disease N.meningitidis and causes endemic and epidemic form of the disease. In the USA the incidence is 0.6-1 per 100 thousand people per year, and this figure may increase in conditions of disease outbreak (see Lieberman et al., 1996, "Safety and Immunogenicity of a Serogroups A/C Neisseria meningitidis Oligosaccharide-Protein Conjugate Vaccine in Young Children", JAMA, 275 , 1499-1503; Schuchat et al., 1997, "Bacterial Meningitis in the United States in 1995," New England J. Med., 337 , 970-976). In developing countries, the frequency of endemic cases significantly higher, and when epidemics, this figure can reach 500 cases per 100 thousand people per year. The mortality rate is very high - about 10-20% in the United States and even higher in developing countries. After the introduction of combination vaccines against Haemophilus influenzae meningococcal disease N.meningitidis becomes the main causative agent of bacterial forms of meningitis in all age groups in the United States (Schuchat et al., 1997, CIT. above).
Based on the parameters of the components of the capsule meningococcus polysaccharide was identified 12 N.meningitidis serogroups. Group a includes the pathogen, which is mainly associated with epidemiological forms zabolevaniya pricheski areas of Africa. Serogroup b and C are associated with the vast majority of cases of meningitis in the United States and most developed countries. Serogroup W135 and Y are associated with the other cases in the United States and developed countries. Currently in meningococcal vaccine is a tetravalent polysaccharide vaccine containing factors serogroups a, C, Y and W135. Being effective in application to adolescents and adults, the vaccine causes a weak immune response and short-term protection, and cannot be applied to young children (see, for example, a weekly report, "Morbidity and Mortality weekly report, Vol. 46, N RR-5, 1997). This is due to the fact that polysaccharides are independent of T-cell antigens, which cause a very weak immune response, which may not be reinforced with (subject to "boosting") by re-immunization. After the success of vaccination against .influenzae were developed with a combination vaccine against serogroups a and C is now to end clinical trials (W.D.Zollinger, "New and Improved Vaccines Against Meningococcal Disease", In "New Generation Vaccines", CIT. above, pp. 469-488; Lieberman et al., 1996, CIT. above; Constantino et al., 1992, "Development and phase I clinical testing of a conjugate vaccine against meningococcus A and C", Vaccine, 10, 691-698).
However, the problem remains serotype In the meningococcus. Currently, this serotype causes about 50% of the total number of cases of meningitis in the USA, Europe and South America. "Polysaccharide" approach cannot be used, because menB capsular polysaccharide is a polymer of linked α(2-8) N-acetylneuraminic acids that are also present in mammalian tissues. It causes tolerance to this antigen: indeed, if we assume the manifestation of the immune response, it will be sent to your own body, i.e. such a response is undesirable. To avoid induction of an autoimmune response and induction of protective immune response is part of the capsule polysaccharide was, for example, chemically modified by substitution of the N-acetyl group on the N-propionyl group, resulting in specific antigenicity remains unchanged (Romero & Outschoorn, 1994, "Current Status of Meningococcal group In vaccine candidates: capsular or non-capsular?", Clin. Environ. Rev., 7 , 559-575).
Alternative approaches to the creation of vaccines against meningitis-In used a complex mixture of proteins of the outer membrane (MRA), including themselves proteins MRA or MRA, enriched porins or deleteregvalue options MRA 4-th class, which are believed to induce the production of antibodies that block the bactericidal activity. In this approach, get the vaccine, full specifications have not yet been received. These vaccines can provide protection against the homologous strain, but this is m are essentially ineffective in cases when there are multiple antigenic variants of outer membrane proteins. To overcome the factor of antigenic variability were obtained multivalent vaccines containing up to 9 different Parinov (see, for example, J..Poolman, 1992, "Development of a meningococcal vaccine", Infect. Agents Dis., 4, 13-28). Other proteins that are used to create "vneshnemembrannye vaccines are proteins OPA and OPC, however, none of the approaches provides not overcome the factor of antigenic variability (see, for example, Ala Aldeen & Borriello, 1996, "The meningococcal transferrin-binding proteins 1 and 2 are both exposed surface and generate bactericidal antibodies capable of killing homologous and heterologous strains", Vaccine, 14, 49-53).
Available are some data on the sequences of meningococcal and gonococcal genes and proteins (for example, in patent applications EP-0467714 and WO 96/29412), but, of course, they are incomplete. Get more data sequences will provide good prospects for identification of secreted or located on the cell surface proteins, which are promising targets for the immune system and which are not characterized antigenic variability. For example, some of the identified proteins could be the components of effective vaccines against meningococcus, some of them could be the components of vaccines against all meningococcal when Latipov and others might be the components of vaccines against all pathogenic forms of genus Neisseriae.
The present invention is proteins comprising amino acid sequences belonging to Neisseria described in the following examples. These sequences belong to N.meningitidis or N. gonorrhoeae.
Also represented proteins comprising sequences that are homologous (i.e. characterized by the identity sequence) amino acid sequences Nasseri shown in the examples. Depending on the specific sequence level of identity is preferably greater than 50% (e.g., 65%, 80%, 90% or more). These homologous proteins include mutants and allelic variants of the sequences described in the examples. Usually 50%or higher identity of the two proteins is considered as evidence of functional equivalence. The level of identity of the two proteins is preferably determined by the method of Smith-Waterman algorithm which is incorporated in a computer MPSRCH program (Oxford Molecular): used the search affine gaps" (i.e. mismatched in two sequences plots) with parameters gap open penalty=12 and gap extension penalty=l".
Further, the present invention is proteins, including fragments of amino acid sequences Nasseri described in the following examples. These fragments must include at least n contiguous amino acids from the base sequence, and depending on the particular sequence, n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more). Preferably, such fragments comprise an epitope of the sequence.
Proteins of the present invention can be obtained, of course, using different approaches (for example, by the methods of recombinant expression, purification from cell culture, chemical synthesis etc) and in various forms (e.g. native, chimeric and so on). Preferably they are obtained in substantially pure or dedicated form (i.e. substantially free from other proteins of Neisseria or cellular proteins of the host body).
In accordance with the following aspect of the present invention provides antibodies that bind to such proteins. It can be polyclonal or monoclonal antibodies that can be obtained with the use of suitable methods.
In accordance with the following aspect of the present invention represented by the nucleic acid comprising the nucleotide sequence of Neisseria described in the examples. In addition, the present invention is a nucleic acid, including homologous sequences (i.e. characterized by the identity of the sequences against nucleotide sequences Nasseri described in the use of the Ah.
Further, the present invention is a nucleic acid that can hybridizing with nucleic acids of Neisseria described in the examples, preferably in the harsh conditions of hybridization (e.g., at 65°in solution 0,1xSSC, 0,5% SDS).
Also provided are nucleic acids, including fragments of such sequences. They should include at least n located consecutive nucleotides from the sequences Nasseri, and depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
In accordance with the following aspect of the present invention is a nucleic acid encoding the proteins and protein fragments of the present invention.
Also it should be clear that the present invention is a nucleic acid comprising sequences complementary to those sequences that were described above (for example, for the purposes of receiving antisense sequences or probes).
Nucleic acids in accordance with the present invention can be, of course, be obtained in many ways (for example, by chemical synthesis, from the libraries of genomic DNA or cDNA directly from the body and so on) and can take different forms (for example, single-stranded, duha the ochechnogo, vector shapes, the shape of the probes and the like).
Additionally it should be noted that the term "nucleic acid" includes DNA and RNA, as well as their analogues, such as those that include modified molecular skeletons, as well as the nucleoproteids (PNA), etc.
In accordance with the following aspect of the present invention represented by vectors comprising the nucleotide sequence of the present invention (for example, expressing vectors) and cell-hosts transformed with such vectors.
In accordance with the following aspect of the present invention represented by the composition containing protein, antibody and (or) nucleic acid in accordance with the present invention. These compositions can be used as, for example, vaccines, or as diagnostic reagents, or as immunogenic compositions.
The present invention is also a nucleic acid, protein or antibody corresponding to the present invention, for use as drugs (such as vaccines) or as diagnostic reagents. It also seems a nucleic acid, protein or antibody in accordance with the present invention in the manufacture of: (1) drugs intended for the treatment or prevention INFI is investing bacteria of the genus Neisseria; (2) a diagnostic reagent intended for detection of the presence of bacteria Neisseria or antibodies specific against them; and / or (3) of the reagent, which may cause the production of antibodies against Neisseria. Mentioned bacteria of the genus Neisseria can be represented by any type or strain (such as N.gonorrhoeae or any N.meningitidis strain, such as strain And strain or strain).
The present invention also provides a method of treating a patient, comprising the introduction of this patient a therapeutically effective amount of nucleic acid, protein and / or antibodies of the present invention.
In accordance with other aspects of the present invention are represented in various ways.
Seems to be a way to obtain proteins of the present invention, comprising the step of culturing the host cell in accordance with the present invention under conditions that promote expression of the protein.
Seems to be a method of obtaining a protein or nucleic acid of the present invention, despite the fact that such a protein or a nucleic acid synthesized in whole or in part, using chemical methods.
It seems the method detection polynucleotides of the present invention, comprising the following steps: (1) contact a nucleotide probe according to the present izobreteny the biological sample under conditions suitable for molecular hybridization to form duplexes; and (2) detection of the above-mentioned duplexes.
Is the method of detection of the proteins of the present invention, comprising the following steps: (1) contact the antibodies of the present invention with the biological sample under conditions suitable for the formation of complex antigen-antibody"; and (2) detection of the mentioned complexes.
The following is an overview of standard methodologies and procedures that can be used to implement the present invention (for example, to use the claimed sequences for vaccination or diagnostic purposes). This review is not a limitation for the present invention, but it is an example of such implementation, which is not strictly necessary.
The practical implementation of the present invention is based, except as specifically stipulated cases, the standard techniques of molecular biology, Microbiology, recombinant DNA and immunology, which are known to specialists in this field of technology. Such techniques are described in detail in the scientific literature: for example, Sambrook, 1989, "Molecular Cloning: A Laboratory Manual", 2d Ed.; "DNA Cloning", Vol. I & II, ed. D.N.Glover, 1985; "Oligonucleotide Synthesis", ed. M.J.Gait, 1984; "Nucleic Acid Hybridization", eds. B.D.Hames & S.J.Higgins, 1984; "reduced and Translation", eds. B.D.Hame & S.J.Higgins, 1984; "Animal Cell Culture", ed. R.I.Freshney, 1986; "Immobilized Cells and Enzymes, IRL Press, 1986; B.Perbal, 1984, A Practical Guide to Molecular Cloning; the series"Methods in Enzymology" (published by Academic Press Inc.), especially volumes 154 and 155; "Gene Transfer Vectors for Mammalian Cells", eds. J.H.Miller & M.P.Calos, Cold Spring Harbor Lab., 1987; "Immunochemical Methods in Cell and Molecular Biology", eds. Mayer & Walker, Acad. Press, London, 1987; Scopes, 1987, "Protein Purification: Principles and Practice, 2d ed., Springer-Verlag, NY; and "Handbook of Experimental Immunology", Vol. I-IV. eds. D.M.Weir & C.C.Blackwell, 1986).
In the present description uses standard abbreviations for designation of amino acids and nucleotides.
All publications, patents and patent applications cited in this text, is included in its full amount in the form of bibliographic references. In particular, this text is included for the information of British patent application No. 9723516.2, 9724190.5, 9724386.9, 9725158.1, 9726147.3, 9800759.4 and 9819016.8.
A composition containing X, "substantially free from Y" when at least 85% by weight of the sum X+Y is the share component of X. Preferably, X is at least about 90% by weight of the total amount of X+Y in the composition, more preferably at least about 95% or even 99% by weight.
The term "including" means "comprising", as well as "contains". For example, a composition "comprising" X may consist exclusively of component X or may include something additional to , for example, the combination of X+Y.
The term "heterologous" refers to two biological components, which are not found together in nature. Such components can be cell-hosts, genes or regulatory segments, such as promoters. Although heterologous components in nature are not detected, they can have joint functionality, for example, when a promoter that is heterologous in relation to gene, functionally connected with him. Another example is a situation in which a sequence of Neisseria is heterologous in relation to the murine host cell. Additional examples can be two epitope of the composition the same or different proteins, which are arranged in a single protein in such a combination, which is never found in nature.
"The starting point of replication" refers to a polynucleotide sequence that initiates and regulates the replication of polynucleotides, such as expressing vector. Point (or website) start replication behaves as an Autonomous unit of polynucleotide replication within the cell, providing the ability to replicate under her control. The presence of the starting point of replication may be necessary to ensure replication of the vector in a specific cell host. If there are multiple start points replication Express the dominant vector can be played in a large number of copies in the presence of suitable proteins inside cells. Examples of start points are Autonomous replication can replicate sequences that are effective in yeast cells, and viral T antigens effective in cell line COS-7.
The term "mutant sequence" defines the DNA, RNA or amino acid sequence that differs from the native or the stated sequence, but having a resemblance to her. Depending on the particular sequence, the level of sequence identity when compared to the native or the stated sequence and the mutant sequence preferably greater than 50% (comprising, for example, 60%, 70%, 80%, 90%, 95%, 99% or more: the calculation is performed using the algorithm of Smith-Waterman described above). In this text, the term "allelic variant" nucleic acid molecule or segment is represented nucleotide sequence is a nucleic acid molecule or segment, which essentially is in the same locus specific genome of another or second isolate, despite the fact that due to natural variability arising from, for example, mutation or recombination processes, characterized by similar, but not identical, to the nucleotide sequence. Encoding the segment allelic variants usually encodes the protein having the output level of physical activity compared with those in the protein, encoded by the gene, which is carried out this comparison. Allelic variant may also include alternating 5'- or 3'-noncoding sections of a particular gene, such as regulatory control segments (see, for example, U.S. patent No. 5753235).
The nucleotide sequence of Neisseria can be expressed using different expression systems: for example, is used for this purpose mammalian cells, baculoviruses, plants, bacteria and yeast.
I. system of mammals
Expression system mammalian known in the art. A mammalian promoter may be any DNA sequence that can bind RNA polymerase mammals, thereby initiating the transcription of downstream (i.e. in the 3'-end) coding sequence (e.g., structural gene) with the formation of mRNA. The promoter must include the site of transcription initiation, which is usually located proximally relative to the 5'-end of the coding sequence, and a TATA box, usually located in the 25-30 nucleotides above the site of transcription initiation. It is believed that the TATA box provides controlled RNA polymerase II start of RNA synthesis in the correct site. The promoter mammals should also include set to enter the promotor element, usually in the 100-200 nucleotides above the TATA box. Top promoter element defines the rate at which transcription is initiated, and can be active in any orientation (Sambrook et al., 1989, "Expression of Cloned Genes in Mammalian Cells", In "Molecular Cloning: A Laboratory Manual", 2d ed.).
Genes of mammalian viruses are typically characterized by intense expressiruemogo and are characterized by a wide range of hosts: therefore, sequences encoding genes of mammalian viruses are particularly promising for use as promoter sequences. Examples include the promoter of the early genes of the virus SV40, promoter LTR of the virus tumors in the mammary gland of the mouse, the main promoter of the late gene of adenovirus (AdMLP) and the promoter simple herpes virus. In addition, the sequence derived from non-viral genes, such as gene metallothionein mouse, are also applicable to the promoter sequence. The expression can be constitutive and regulated (inducible)that depends on induction of the promoter by glucocorticoids in cells controlled by these hormones.
The presence of the enhancer element (enhancer) together with promoter elements described above, usually leads to increased levels of expression. Enhancer is a regulatory DNA sequence that str is found to stimulate transcription of up to a thousand times the level in the case of its binding to homologous or heterologous promoters, despite the fact that the synthesis begins in the normal originating site RNA. Enhancers are also active when they are placed either above or below the site of transcription initiation, in both normal and inverted by the switching mechanism of the phase orientation, or at a distance even greater than 1000 nucleotides from the promoter (Maniatis et al., 1987, Science, 236, 1237; Alberts et al., 1989, "Molecular Biology of the Cell, 2d ed.). In particular, can be used enhancer elements derived from viral genomes, because they are usually characterized by a wide range of potential hosts. Examples are early enhancer gene of SV40 virus (Dijkema et al., 1985, EMBO J. 4, 761) and enhancer + promoter derived from the plot of the long terminal repeats (LTR) of the rous sarcoma virus (Gorman et al., 1982b, Proc. Natl. Acad. Sci. USA, 79, 6777) and human cytomegalovirus (Boshart et al., 1985, Cell 41, 521). In addition, some enhancers are adjustable and become active only in the presence of inducer, such as a hormone or metal ion (Sassone-Corsi & Borelli, 1986, Trends Genet., 2, 215; Maniatis et al., 1987, Science, 236, 1237).
The DNA molecule can be expressed within mammalian cells. The promoter sequence may be directly attached to the DNA molecule taking into account the fact that the first N-end amino acid in the recombinant protein should always be a methionine, which is encoded by the start-codon ATG. If chelation is) N-terminal part can be derived from the protein by incubation in vitro with cyanogenmod.
On the other hand, foreign proteins can also secretariats of cells directly in the culture medium as a result of creating chimeric DNA molecules that encode a chimeric protein comprising a leader sequence segment that provides secretion of the foreign protein in mammalian cells. Preferred is the presence of processing sites encoded between the leader segment and alien genome, which could be cleaved either in vivo or in vitro. Leader segment typically encodes a signal peptide comprised of hydrophobic amino acids, which provide for secretion of the protein from the cell. Adenovirus tripartite leader segment is an example of a leader sequence that provides secretion of the foreign protein by mammalian cells.
Usually the termination of transcription and polyadenylation sequence recognized by the mammalian cells are regulatory segments, arranged in 3'-end from the stop codon, i.e. together with promoter elements, they are motifs flanking the coding sequence. the 3'end of the Mature mRNA is formed by site-specific post transcriptional cleavage and polyadenylation (Birnstiel et al., 1985, Cell, 41, 49; Proudfoot & Whitelaw, 1988, "Termination and 3' end processing of eukaryotic RNA", In "reduced and splicing", ed. B.D.Hames & D.M.Glover; Proudfoot, 1989, Trends Biochem. Sci., 14, 105). These sequences provide the transcription of mRNA, which can then be translated into the polypeptide encoded by the original DNA. Examples of signals termination of transcription and polyadenylation are those motives that originate from the genome of SV40 virus (Sambrook et al., 1989, "Expression of cloned genes in cultured mammalian cells", In "Molecular Cloning: A Laboratory Manual").
Usually the above-described components, including the promoter, polyadenylate signal and the site of transcription termination include simultaneously the composition of expressing designs. Enhancers, introns, including functional donor and acceptor splicing sites, and leader sequence can also be included in expressing the design, if this is desirable. Expressing designs are often supported in the form of a replicon, such as an extrachromosomal element (e.g., plasmids)capable of stable to persist in the host organism, such as a mammal cells or bacteria. The replication system of mammals include those systems that are derived from animal viruses to replicate together with transregulatory factors. For example, plasmids, including replication system papovaviruses, still is how the monkey virus SV40 (Gluzman, 1981, Cell, 23, 175), or polyomaviruses replicated in an exceptionally large number of copies in the presence of the appropriate viral T antigen. Additional examples of replicons for mammalian cells are those that result from bovine papillomavirus and Epstein-Barr. In addition, the replicon can carry two replication systems, thus provides the ability to maintain them, for example, in mammalian cells for the purpose of expression in prokaryotic cells to clone and amplificatoare. Examples of such bifunctional vectors for mammalian/bacterial include design pmt2 (Kaufman et al., 1989, Mol. Cell. Biol., 9, 946) and pHEBO (Shimizu et al., 1986, Mol. Cell. Biol., 6, 1074).
The procedures selected transformation depends on the type of the host body, which will be transformed. Methods of application of heterologous polynucleotides into mammalian cells are well known in the art: they include mediated by dextran transfection, the precipitation of calcium phosphate mediated polybrene transfection, fusion of protoplasts, electroporation, encapsulation of polynucleotide(s) in liposomes, and direct microinjection DNA in the nucleus of target cells.
Lines of mammalian cells suitable as hosts for the purposes of expression, are well known and include a large number immortali avannah cell lines, available from the American type culture collection (ATSC), including, but not limited to, cells of the Chinese hamster ovary (Cho)cells, HeLa human cells of the kidneys of newborn hamsters (KSS), the cells of green monkey kidney (COS)cells, hepatocellular carcinoma person (for example, HepG2) and many other cell lines.
2. Baculovirus system
Polynucleotide encoding the protein can also be embedded in suitable expressing vector for insect cells: functional way connected with regulatory elements within that vector. When designing vector uses techniques that are well known in the art. In General, the components of the expression system include the actual vector for transfer, usually a bacterial plasmid, which includes a fragment of the baculovirus genome, and a standard restriction site that is designed to embed a heterologous gene or genes that will be expressed; a wild type baculovirus, characterized by sequence similarity with baculoviruses fragment of the vector to transfer (this ensures that homologous recombination of heterologous gene into the baculovirus genome); and the appropriate cells of the host insect and cultural environment.
After making the follower of the spine of the DNA, encodes a specific protein, the vector for the transfer of this vector and the viral genome of the wild type used for transfection in a cell of the host insect, in which the vector and the viral genome can recombine. Packed recombinant virus is expressed, and the recombinant plaques can be identified and cleared. Materials and methods formation of gene-expression systems baculovirus/insect cells available in the form of special kits on a commercial basis, such as, inter alia, on the company Invitrogen (San Diego, USA): a set of reagents "Mahvash". These techniques generally known to experts in the art and fully described in the manual summers and Smith (Summers & Smith, 1987, Texas Agricult. .. Stat. Bull., N 1555) (hereinafter cited as "Summers & Smith").
Before embedding the DNA sequence that encodes a protein, the composition of the baculovirus genome described above components, including the promoter, leader segment (if it is desirable)of interest coding sequence and the site of transcription termination, usually assemble in the intermediate structure (a vector for transfer). Such construction may include in its composition a single gene and functionally attached to regulatory elements; or multiple genes, each of which has "its own" n the boron functionally attached regulatory elements; or multiple genes under the control of the same regulatory elements. Intermediate alternating structures are often supported in the form of a replicon, such as an extrachromosomal element (e.g., a plasmid), can stably be maintained in the body of the host, such as bacteria. This replicon should include a replication system, thereby maintaining a replication in a suitable organism, the host, with the aim of cloning and amplificatoare.
Currently, the most common vector for transfer with a view to the introduction of foreign genes into AcNPV is RS. Can also be formed and many other vectors known to specialists in this field of technology. They include, for example, pVL985 (which changes the start-codon of the gene polyhedrin from ATG to ATT, and which introduces a BamHI cloning site 32 pairs of nucleotides below codon ATT; see Luckow &Summers, 1989, Virology, 17, 31.
Commonly used plasmid also includes the polyadenylation signal of the gene polyhedrin (Miller et al., 1988, Ann. Rev. Environ., 42, 177) and prokaryotic gene for resistance to ampicillin (amp) and the replication origin, required for selection and reproduction in E. coli cells.
Baculovirus transfection vectors typically include baculovirus promoter. Baculovirus promoter is any sequence the DNA, the ability to communicate with baculovirus RNA polymerase and initiate transcription of the coding sequence (e.g., structural gene) in the direction 5'-3' with the formation of mRNA. The promoter must include the site of transcription initiation, which is usually placed proximally relative to the 5'-end of the coding sequence. This site of transcription initiation usually involves the binding site of RNA polymerase and the actual site of transcription initiation. Baculovirus transfection vector may also include a second domain, defined as "enhancer", which, if present, is usually located distal to the structural gene. The expression can be either inducible or constitutive.
Structural genes, intensely transcribed at late stages of the viral infectious cycle, allow you to select specifically applicable to the promoter sequence. Examples are sequences derived from the gene encoding a viral protein - polyhedrin (Friesen et al., 1986, "The Regulation of Baculovirus Gene Expression", In "The Molecular Biology of Baculoviruses, ed. W.Doerfler; European patent publication No. 127839 and 155476), and the gene encoding the protein p10 (Vlak et al., 1988, J. Gen. Virol., 69, 765).
DNA encoding suitable signal sequences can be derived from genes encoding secreted proteins insects or baculovir the owls, such as gene polyhedrin baculovirus (Carbonell et al., 1988, Gene, 73, 409). On the other hand, when the signals of posttranslational modifications in mammalian cells (such as cleavage of the signal segment, proteolytic cleavage, and phosphorylation), apparently, recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conservative in the cells of vertebrates and invertebrates, to ensure secretion in insects can also be used leader segments, not related to the origin of the insects, such as those that are derived from genes encoding α-interferon person (Maeda et al., 1985, Nature, 315, 592), the releasing-factor gastrin person (Lebacq-Verheyden et al., 1988, Mol. Cell. Biol., 8, 3129), interleukin-2 (Smith et al., 1985, Proc. Natl. Acad. Sci. USA, 82, 8404), interleukin-3 mouse (Miyajima et al., 1987, Gene, 58, 273) and glucocerebrosidase man (Martin et al., 1988, DNA 7, 99).
Recombinant polypeptide or polyprotein can be expressed intracellularly or, if it is expressed with the participation of the special regulatory sequences, it can secretariats. Efficient intracellular expression Nehemiah carbon proteins usually requires a heterologous genes, which ideally include a short leader sequence, including eligible which signals the initiation of translation, pre-start-codon ATG. If desirable, the methionine residue from N-Terminus can be derived from the Mature protein by incubation in vitro with cyanogenmod.
On the other hand, recombinant polyprotein or proteins that are not natively sekretiruemyi, can be secreted from insect cells by creating chimeric DNA molecules that encode a chimeric protein comprising a leader segment that provides secretion of the protein, alien insects. Segment leader sequence typically encodes a signal peptide comprised of hydrophobic amino acids, which controls the movement of the protein in the endoplasmic reticulum.
After embedding the DNA sequence and (or) gene encoding the expression product, which is a precursor of a specific protein, a cell of the host insect at the same time transform the heterologous DNA vectors for transfer and genomic DNA of wild-type baculovirus - usually used cotransfection methods. The promoter and the termination sequence of the transcription of this design usually must include the segment of the baculovirus genome length 2-5 thousand pairs of nucleotides. Methods of application of heterologous DNA at the desired site in the structure of baculovirus known in the art (see Summers & Smith, CIT. above; Ju et al., 1987; Smith et al., 983, Mol. Cell. Biol., 3, 2156; Luckow &Summers, 1989). For example, the embedding can be performed inside a gene, such as gene polyhedrin, which is homologous double recombination; embedding can be carried out by restriction enzyme site, an artificially created within desirable baculovirus gene (Miller et al., 1989, BioEssays, 4, 91). The DNA sequence when it is cloned into a gene polyhedrin expressing vector, planiruetsja on both sides (5' and 3'sequences, characteristic gene polyhedrin, and is placed below polietilenovogo promoter.
Newly formed baculovirus expressing vector sequentially packaged into infectious recombinant baculovirus. Homologous recombination occurs with a low frequency (from about 1% to about 5%), therefore, the vast majority produced by cotransplantation viruses represents the wild-type virus. This means that a special approach is necessary to identify recombinant viruses. The advantage of expressing this system is the possibility of visual screening, identifying recombinant viruses. Protein polyhedrin characteristic of natural viruses produced in very large quantities in the nuclei of infected cells at late the phases of the cycle of viral infection. Accumulated protein polyhedrin forms included the Taurus, which also contain embedded particles in them. These included calf size up to 15 microns cause significant refraction of light: it gives them a bright glow that can be easily rendered in normal light microscope. In cells infected with recombinant virus included bullock no. To identify recombinant viruses and viruses of wild-type transfection supernatant layer contribute to monoclonal culture of insect cells using techniques well known to specialists in this field of technology. Namely plaques are subjected to analysis under a light microscope to identify the presence (as a characteristic of the wild-type virus) or absence (a sign of recombinant virus) included Taurus ("Current Protocols in Microbiology", Vol. 2, eds. Ausubel et al., section 16.8 (Supp. 10, 1990); Summers & Smith, CIT. above; Miller et al., 1989).
Recombinant baculovirus expressing vectors were constructed with the purpose of infection in different types of insect cells. For example, without limitation, recombinant baculoviruses were generated for mosquito Aedes aegypti, scoops Autographa californica, the silkworm Bombyx mori, Drosophila Drosophila melanogaster, Spodoptera frugiperda, and scoops Trichoplusia ni (patent application WO 89/046699; Carbonell et al., 1985, J. Virol., 56, 153; Wright, 1986, Nature, 321, 718; Smith et al., 1983, Mol. Cell. Biol. 3, 2156; as an overview, see Fraser et al., 1989, In Vitro Cell. Develop. Biol., 25, 225).
Cells and culture medium are available on a commercial basis as to direct and chimeric expression of heterologous polypeptides in baculovirus expressing the system; the technology of culturing cells in General are well known to experts in the art (see, e.g., Summers & Smith, CIT. above).
Modified insect cells can then be grown in an appropriate nutrient medium, which will ensure stable maintenance of plasmids present in the modified insect host. When expressing the product is under the control of the inducible regulators, master-organism can be grown at very high densities with induction of expression. On the other hand, when the expression is constitutive, the desired product will be constantly expressed in the culture medium and culture medium may continuously circulate - this allows you to select the desired interest product and replenish depleted nutrients. This product can be purified using techniques such as chromatography, such as HPLC, affinity chromatography, ionoobmennaya chromatography and the like; electrophoresis; centrifugation in density gradient; extraction from a solution or similar. If you think the mo, this product can be subjected to additional cleaning to ensure substantial removal of any insect proteins, which are also sekretiruemyi in culture medium or formed as a result of lysis of insect cells, resulting in a product that is essentially free from debris hosts, such as proteins, lipids and polysaccharides.
To ensure expression of the protein of the recombinant cell host, derived from the obtained transformants, incubated under conditions that provide for expression of the sequence encoding the recombinant protein. These conditions may vary depending on the selected host cell. However, such conditions can be easily selected by a person skilled in the field of technology on the basis of known scientific data.
3. Herbal system
In the art knows numerous genetic expression systems based on plant cells and whole plants. Examples of such genetic plant expression systems can be found in U.S. patent No. 5693506, 5659122 and 5608143. Additional examples of genetic expression in cultured plant cells described by the Valuation (Zenk, 1991, Phytochemistry, 30, 3861-3863). Description of the signal peptides of plant proteins can be found, in addition to the to the above sources, in the works Vaulcombe et al., 1987, Mol. Gen. Genet., 209, 33-40; Chandler et al., 1984, Plant Mol. Biol., 3, 407-418; Rogers, 1985, J. Biol. Chem., 260, 3731-3738; Rothstein et al., 1987, Gene, 55, 353-356; Whittier et al., 1987, Nucl. Acids Res., 15, 2515-2535; Wirsel et al., 1989, Mol. Environ., 3, 3-14; Yu et al., 1992, Gene, 122, 247-253. Description of the parameters of the regulation of plant gene expression involving hormones, gibberellic acid and secreted enzymes induced by gibberellic acid, can be found in the monograph R.L.Jones & J.MacMillin, 1984, "Gibberellins", In "Advanced Plant discrimination", ed. M.B. Wilkins, Pitman Publ. Ltd, London, pp. 21-52. Materials describing other genes regulated involving metabolic mechanisms: Sheen, 1990, Plant Cell 2, 1027-1038; Maas et al., 1990, EMBO J. 9, 3447-3452; Benkel & Hickey, 1987, Proc. Natl. Acad. Sci. USA, 84, 1337-1339.
Usually using methodologies known in the art, the desired polynucleotide sequence is embedded in the structure of the expression cassettes, including genetic regulatory elements, designed for operation in plants. The expression cassette was built in desirable expressing vector with sequences that are located above and below the expression cassette suitable for the expression in the plant host. Related sequences can be plasmid or viral origin and provide necessary characteristics to the vector, which will provide vectors the ability to endure the ü DNA from the source organism, the host, used for cloning, such as a bacterium, in a desirable host-plants. Base bifunctional ("bacterial-plant") vector design preferably should provide a wide host range to include a prokaryotic replication origin prokaryotic selective marker and, in the variant with the transformation of the bacteria Agrobacterium, the sequence T-DNA, which will allow the transfer of genes based on the transformation using Agrobacterium. When identification of the heterologous gene is difficult, this design should preferably also include a selective marker, a method to identify whether the transformed plant cell. Review by selective markers, for example, members of the grass family, can be found in the article Wilmink & Dons, 1993, Plant Mol. Biol. Reptr., 11(2):65-185.
Sequence suitable for ensuring the integration of the heterologous sequence into the genome of plants, is also recommended. They may include transposon sequences and the like, which are connected with the processes of homologous recombination, as well as Ti-sequences, which cause random embedding heterologous expression cassette into the plant genome. Suitable prokaryotic selective markers include signs of resistance to antibiotics, so it is to ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be included in the vector, as known in the art.
The nucleic acid molecule of the present invention can be incorporated into expression cassettes, designed to ensure the expression of the protein of interest (protein). Usually use a single expression cassette, although it is possible to use two or more such tapes. Recombinant expression cassette should include in addition to the sequence that encodes a heterologous protein, the following elements: promoter segment, 5'-noncoding sequences of the plant genome, the initiating codon, the presence or absence of which depends on whether it is part of the analyzed structural gene, and the sites of termination of transcription and translation. Unique recognition sites by restrictase 5'- and 3'-ends of the tapes provide a convenient incorporation into the composition of the pre-existing vector.
Heterologous coding sequence may be related to any protein of the present invention. Sequence encoding a protein of interest, will encode the signal segment, which will provide the processing and movement protein, if necessary, and generally must be devoid of any sequence, which could cause binding of the desired protein of the present invention with a membrane. Despite the fact that in most cases the site of transcription initiation is designed for the gene that is expressed and translocases in the germination process, by use of the signal segment, providing the movement, it is also possible to provide the movement of the desired protein. In this case, the protein of interest (protein) will be transferred from the cells in which it was expressed, therefore, it can be effectively collected. Usually the secretion of seeds is provided from the aleurone region or didovoho epithelium in the endosperm of the seed. Although there is no specific need to secretion of the protein from the cells in which it was developed, this approach facilitates the isolation and purification of the recombinant protein.
As the final expression of the desired gene product must occur in eukaryotic cells, it is desirable to determine whether any of the sites of the cloned gene sequences that could be lost in the form of introns under the action of the splicing mechanism, the body's own master. If they are, then using the method of directed (site-specific) mutagenesis such "intron" Uch the flow may be modified to avoid the loss of genetic material due to the presence of londontrini signals (Reed & Maniatis, 1985, Cell 41, 95-105).
The vector may be microinjection in plant cells using micropipets that will provide a mechanical transfer of recombinant DNA (Crossway, 1985, Mol. Gen. Genet., 202, 179-185). Genetic material may be transferred into the plant cell using polyethylene glycol (Krens et al., 1982, Nature, 296, 72-74). Another way of making segments of nucleic acids is a high-speed ballistic introduction of fine particles ("shells"), inside or on the surface of which is a nucleic acid (Klein et al., 1987, Nature, 327, 70-73; Knudsen & Muller, 1991, Planta, 185, 330-336 - in this work describes the application of the method of bombarding particles of the endosperm of barley in order to obtain transgenic barley plants). Another way of making can be fusion of protoplasts with other components, such as mini-cells, cells, complementary mechanism or other able to participate in fusion with protoplasts of Taurus, with the lipid surface (Fraley et al., 1982, Proc. Natl. Acad. Sci. USA, 79, 1859-1863).
The vector may also be introduced into plant cells using electroporation (Fromm et al., 1985, Proc. Natl. Acad. Sci. USA 82, 5824). In this technique, plant protoplasts are subjected to electroporation in the presence of plasmids comprising genetic design is the construction. Electrical impulses, characterized by a high value of electric field intensity, reversible "hole" biological membranes, thereby penetration of plasmids inside. Elektrooborudovanie plant protoplasts regenerate their cell wall, share and form celluci.
All plants from which can be isolated protoplasts and subsequent cultivation and obtaining whole regenerated plants can be transformed in accordance with the present invention so as to produce whole plants carrying the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major types of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants, for example, are species of the genera Fragaria (strawberry), Lotus (Lotus), Medicago (alfalfa), Onobrychis (sainfoin), Trifolium (clover), Trigonella (fenugreek), Vigna (cowpea), Citrus (citrus), Linum (flax), Geranium (geranium), Manihot (cassava), Daucus (carrot), Arabidopsis (rezunaweku), Brassica (cabbage, rutabaga, turnip), Raphanus (radish), Sinapis (mustard), Atropa (deadly nightshade-belladonna), Capsicum (sweet pepper), Datura (Datura), Hyoscyamus (henbane), Lycopersion (tomato), Nicotiana (tobacco), Solanum (Basle is), Petunia (Petunia), Digitalis (weed), Majorana (marjoram), Cichorium (chicory), Helianthus (sunflower), Lactuca (lettuce), Bromus (brome), Asparagus (asparagus), Antirrhinum (snapdragons), Hererocallis, Nemesia, Pelargonium (pelargonium), Panicum (millet), Pennisetum (American millet), Ranunculus (Buttercup), Senecio (Senecio), Salpiglossis, Cucumis (cucumber, melon), Browaalia, Glycine (soybean), Lolium (ryegrass), Zea (maize), Triticum (wheat), Sorghum (sorghum) and Datura.
Methods of regeneration varies depending on the plant species, but in General, always beginning a suspension of transformed protoplasts carrying copies of the heterologous gene. Then form kaluzny tissue, which shoots can be induced with the subsequent formation of roots. On the other hand, in the suspension of protoplasts can be induced formation of embryos. These embryos germinate as normal embryos, forming plants. Culture medium should generally contain various amino acids and hormones, such as auxin and cytokines. Also preferred is the addition of glutamic acid and Proline in the culture medium, especially for such species as corn and alfalfa. Shoots and roots in normal developing at the same time. Regeneration efficiency will depend on the cultural environment, genotype and on the parameters of a particular culture. If these three variables are controlled, the regeneration is vos is proizvodimoj and repeatable.
In some systems, the cultivation of plant cells is desired protein of the present invention may stand alone or, on the other hand, this protein can be extracted from whole plants. In those cases, when the desired protein of the present invention is secreted into the culture medium, it can be assembled. On the other hand, embryos and policemen" with remote embryonic part or other plant tissue can be mechanically crushed to identify secreted protein from cells and tissues. The resulting mixture can be suspended in a buffer solution with the aim of re-extraction of soluble proteins. Standard methods for isolation and purification of proteins can be used for purification of recombinant protein. The parameters of time, temperature, pH, oxygen content and volume can be increased by using routine procedures to optimize expression and allocation of the heterologous protein.
4. Bacterial systems
Methods expression in bacterial cells are well known. Bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating transcription of downstream (in the direction 3') coding sequence (e.g., structural gene) with the formation of MRR is. The promoter must be present on the site of transcription initiation, which usually include a proximally relative to the 5'-end of the coding sequence. This site of transcription initiation usually involves the binding site of RNA polymerase and the actual site of transcription initiation. Bacterial promoter may also include a second domain, called the "operator", which may overlap with neighboring binding site of RNA polymerase, which begins the synthesis of RNA. The operator provides negatively regulated (i.e. inducible) transcription: gene-repressing protein can bind to the operator, thereby inhibiting the transcription of a specific gene. Constitutive expression can occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be secured by threading sequence, gene-activating protein, which is: if present, is located proximally (with 5'-end) in relation to the binding site of RNA polymerase. An example of gene-activating protein is a catabolic activator (RAA), who is the assistant to the initiation of transcription of the lac operon in the genome of Escherichia coli Escherichia coli (E. coli) (Raibaud et al., 1984, Annu. Rev. Genet., 18, 173). Thus, regulation of expression can be negative or positive, thereby increasing the sludge is weakening transcription.
The sequence encoding the enzymes of the metabolic pathways that are specifically applicable to the promoter sequence. Examples include promoter sequences derived from genes encoding enzymes involved in the metabolism of sugars, such as galactose, lactose (lac) (Chang et al., 1977, Nature 198, 1056), and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as the enzymes of the biosynthesis of tryptophan (trp) (Goeddel et al., 1980, Nucl. Acids Res., 8, 4057; Yelverton et al., 1981, Nucl. Acids Res., 9, 731; U.S. patent No. 4738921; European patent application EP-0036776 and ER AND-0121775). Promotor system g-lactamase (bla) (Weissmann, 1981, "The cloning of interferon and other mistakes", In "Interferon 3", ed. I.Gresser) and promotor system PL λ-bacteriophage (Shimatake et al., 1981, Nature, 292, 128) and phage T5 (U.S. patent No. 4689406) can also be used as promoter sequences.
In addition, synthetic promoters that are not found in nature, can also function as a bacterial promoters. For example, the sequence of activation of transcription of one of the bacterial or bacteriophobic promoters can be connected with operandii sequences of other bacterial or bacteriophagous promoter, resulting in a synthetic hybrid promoter (patent When And No. 4551433). For example, the tac promoter is a hybrid promoter, a trp-lac, consisting of sequences of the trp promoter and lac promoter under control of the lac repressor (Amann et al., 1983, Gene, 25, 167; de Boer et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 21).
Next, the bacterial promoter may include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. Naturally occurring promoter non-bacterial origin can also be connected to a compatible RNA polymerase with high-level expression of some genes in prokaryotic cells. System "RNA polymerase/promoter of bacteriophage T7 is an example of a combined promoter system (Studier et al., 1986, J. Mol. Biol., 189, 113; Tabor et al., 1985, Proc. Natl. Acad. Sci. USA, 82, 1074). In addition, a hybrid promoter can also consist of bacteriophagous promoter and operator segment E.coli (EPO-And-0267851).
In addition to functional promoter sequence, the efficient binding site on the ribosome is also applicable to ensure the expression of foreign genes in prokaryotic cells. In E.coli the binding site on the ribosome called the Shine-dalgarno sequence (SD): it includes the start codon ATG and a sequence 3-9 nucleotides located at 3-11 well is leotide above from the start codon (Shine et al., 1975, Nature, 254, 34). The SD sequence is believed, contributes to the binding of mRNA to the ribosome by mating grounds in the structure of the SD sequence and the 3'-end of 16S-pPHK E.coli (Steitz et al., 1979, "Genetic signals and nucleotide sequences in messenger RNA", In Biological Regulation and Development: Gene Expression, ed. R.F.Goldberger). For the expression of eukaryotic genes and prokaryotic genes used "weak" binding site on the ribosome (Sambrook et al., 1989, "Expression of cloned genes in Escherichia coli", In "Molecular Cloning: A Laboratory Manual").
The DNA molecule can be expressed intracellularly. The promoter sequence may be directly attached to the DNA molecule: in this case the first amino acid from the N-end must always be a methionine, which is encoded by the start-codon ATG. If desirable, the methionine from the N-Terminus can be derived from the protein by incubation in vitro with cyanogenmod or by incubation in vivo or in vitro with bacterial enzyme, N-terminal methioninamide (European patent application EPO-And-0219237).
Chimeric proteins are an alternative way of controlling expression. Typically, the DNA sequence encoding the N-terminal site of the endogenous bacterial protein, or other stable protein, is drained from the 5'end of heterologous coding sequences. The result of the expression of such a construction leads to the formation of chimeras (hybrid) two aminoxy the pilot sequences. For example, the cellular gene of bacteriophage λ can be connected at its 5'-end with the alien gene and subsequent expression in bacteria. The resulting chimeric protein preferably retains its structure in the website for processing with the participation of the enzyme (factor XA), resulting in cleavage of phage protein from the product of the foreign gene (Nagai et al., 1984, Nature, 309, 810). Chimeric proteins can also be generated using sequences of genes lacZ (Jia et al., 1987, Gene, 60, 197), trpE (Allen et al., 1987, J. Biotechnol., 5, 93; Makoff et al., 1989, J. Gen. Environ., 135, 11) and Chey (European patent application EP-A-0324647). The DNA sequence in the region of connection of two amino acid sequences can encode a cleavage site, and may not encode it. Another example is a chimeric protein ubicacin. Such a chimeric protein is formed in such a way that within itself ubiqitious segment preferably has kept the site a target for enzyme processing (for example, specific processing ubicacin protease)required for removal of ubicacin from a foreign protein. Using this approach can be selected native alien proteins (Miller et al., 1989, Bio/Technol., 7, 698).
On the other hand, foreign proteins can also be secreted by cells as a result of creating chimeric DNA molecules that b which encodes a chimeric protein, comprising a peptide fragment of the signal sequence that provides secretion of the foreign protein by the bacterial cells (U.S. patent No. 4336336). The fragment of the signal sequence is typically encodes a signal peptide comprised of hydrophobic amino acids, which provide for secretion of the protein from the cell. This protein is secreted or in the culture medium (in the variants with gram-positive bacteria)or periplasmatic the space between the inner and outer membranes of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro in the area between the fragment of the signal peptide and of the foreign gene product.
DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the gene of outer membrane protein gene (ompA) (Masui et al., 1983, In "Experimental Manipulation of Gene Expression"; Ghrayeb et al., 1984, EMBO J. 3, 2437) and signal sequence of the phoA gene encoding alkaline phosphatase of E. coli (Oka et al., 1985, Proc. Natl. Acad. Sci. USA, 82, 7212). As an additional example, the signal sequence of the gene α-amylase different strains of Bacillus, the genus Bacillus, which can be used to ensure the secretion of heterologous proteins from the notches hay Bacillus B.subtilis (Palva et al., 1982, Proc. Natl. Acad. Sci. USA, 79, 5582; European patent application EP-A-0244042).
Usually the sequence termination of transcription recognized by bacteria, presents regulatory segments that are 3'-end from the stop codon, respectively, along with the promoter they flank the particular coding sequence. These sequences provide the transcription of mRNA, which can then be translated into the polypeptide encoded by this DNA. Sequence termination of transcription often include DNA sequences consisting of about 50 nucleotides, capable of forming vypechennye patterns, which provide termination of transcription. Examples are sequences termination of transcription derived from genes with strong promoters such as the trp gene of E. coli, as well as other biosynthetic genes.
Usually the above-described components, including the promoter, signal sequence (if desired), consider the coding sequence and the sequence termination of transcription are embedded together in expressing the design. Expressing design often supported in part replicon, such as an extrachromosomal element (e.g., a plasmid), can stably be maintained in the body of the host, such as bacteria the Oia. This replicon should include a replication system that would provide him the ability to persist in a prokaryotic organism, intended either for expression or for cloning and amplification. In addition, the replicon can be either vysokonapornoj or miscompiles the plasmid. Vysokogorya plasmid should, in principle, be characterized by the number of copies of from about 5 to about 200, and usually from about 10 to about 150. The body is the master, bearing vysokomobilnuju plasmid, preferably should bear at least about 10 plasmids, and more preferably at least about 20 plasmids. And vysokoopasnye, and nicocodine plasmids can be selected on the basis of the influence of the vector and the foreign protein in the body is the master.
On the other hand, expressing constructs can be integrated into the bacterial genome using an integration vector. Integration vectors typically includes in its structure at least one sequence which is homologous to the site of bacterial chromosomes: this ensures that the vector is able to integrate. As is, the integration is due to the recombination between homologous DNA in the vector and the bacterial chromosome. For example, the integration vector designed the haunted on the basis of DNA of different strains of Bacillus, integrated into the chromosome of Bacillus (European patent application EP-A-0127328). Integration vectors can also include phage or transposon sequence.
Usually extrachromosomal and integrated expressing constructs may include selective markers, providing selection of transformed bacterial strains. Selective markers can be expressed in bacterial organism, the host, and may include genes that make bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies et al., 1978, Annu. Rev. Environ., 32, 469). Selective markers can also include biosynthetic genes, such as genes for enzymes involved in the biosynthesis of histidine, tryptophan and leucine.
On the other hand, some of the above components can be incorporated together with each other in the transformation vectors. Transformation vectors typically include a selective marker that is either stored in the replicon, or converted into an integrating vector as described above.
Expressing and transforming the vectors (either extrachromosomal or integrating vectors) were designed to transform many kinds of bacteria. For example, expressing vectors were created, which beside other things, for the following bacterial species: Bacillus subtilis Bacillus subtilis (Palva et al., 1982, Proc. Natl. Acad. Sci. USA, 79, 5582; European patent application EP-A-0036259 and EP-A-0063953; international patent application WO 84/04541), Escherichia coli Escherichia coli (Shimatake et al., 1981, Nature, 292, 128; Amann et al., 1985, Gene, 40, 183; Studier et al., 1986, J. Mol. Biol., 189, 113; European patent application EP-A-0036776, EP-A-0136829 and EP-A-0136907), Streptococcus Streptococcus cremoris (Powell et al., 1988, Appl. Environ. Environ., 54, 655), Streptococcus lividans (Powell et al., 1988, Appl. Environ. Environ., 54, 655), Streptomyces Streptomyces lividans (U.S. patent No. 4745056).
Methods of introducing exogenous DNA into a bacterial organisms-the hosts are well known in science and usually involve the transformation of bacteria treated with calcium chloride or other agents, such as divalent cations and dimethylsulfoxide. DNA can also be introduced into bacterial cells by electroporation method. Transformation procedures usually vary depending on the type of bacteria that will transform: see, for example, Masson et al., 1989, FEMS Environ. Lett., 60, 273; Palva et al., 1982, Proc. Natl. Acad. Sci. USA, 79, 5582; European patent application EP-A-0036259 and EP-A-0063953; international patent application WO 84/04541 (bacteria of the genus Bacillus), Miller et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 856; Wang et al., 1990, J. Bacteriol., 172, 949 (bacteria of the genus Campylobacter), Cohen et al., 1973, Proc. Natl. Acad. Sci. USA, 69, 2110; Dower et al., 1988, Nucl. Acids Res., 16, 6127; Kushner, 1978, "An improved method for transformation of Escherichia coli with ColE1-derived plasmids", In "Genetic Engineerin: Proc. Intern. Symp. on Genet. Engineering", eds. H.W.Boyer & S.Nicosia; Mandel et al., 1970, J. Mol. Biol., 53. 159; Which Deletes An Object, 1988, Biochim. Biophys. Acta, 949, 318 (Escherichia), Chassy et al., 1987, FEMS Environ. Lett., 44, 173 (dairy bacteria of the genus Lactobacillus), Fiedler et al., 1988, Anal. Biochem., 170, 38 (found in the genus Pseudomonas), Augustin et al., 1990, FEMS Environ. Lett., 66, 203 (bacteria of the genus Staphylococcus), Barany et al., 1980, J. Bacteriol., 144, 698; Harlander, 1987, "Transformation of Streptococcus lactis by electroporation", In Streptococcal Genetics, eds. J.Ferretti & R.Curtiss III; Perry et al., 1981, Infect. Immunol., 32, 1295; Powell et al., 1988, Appl. Environ. Environ., 54, 655; Somkuti et al., 1987, Proc. 4th European Congr. Biotechnol., 1, 412 (bacteria of the genus Streptococcus).
5. Expression in yeast
Yeast expressing the system is also well-known to specialists in this field of technology. Yeast promoter is any DNA sequence capable of connecting the yeast RNA polymerase and initiate transcription of the downstream (3'-end) coding sequence (e.g., structural gene) with the formation of mRNA. The promoter must include the site of transcription initiation, which is usually placed proximally relative to the 5'-end of the coding sequence. This site of transcription initiation usually involves the binding site of RNA polymerase (i.e. the "TATA box") and the actual site of transcription initiation. Yeast promoter may also include a second domain, called "upper activator sequence (UAS), which, when present, usually raspolagaetsja distal with respect to the structural gene. The UAS segment provides a regulated (inducible) expression. Constitutive expression occurs in the absence of UAS segment. Regulated expression can be either positive or negative, thereby either strengthens or weakens the transcription.
Yeast is an organism-fermenter, characterized by active metabolism: therefore, the sequence encoding the enzymes of metabolic pathways, represent a particularly promising promoter sequence. Examples are alcoholdehydrogenase (ADH) (European patent application EP-A-0284044), enolase, glucokinase, glucose-6-fortismere, glyceraldehyde-3-phosphatedehydrogenase (GAP or GAPDH), glucokinase, phosphofructokinase, 3-phosphoglycerate and piruwatkinaza (Hands) (European patent application EPO-And-0329203). Yeast gene RNA, encoding acid phosphatase, also is applicable to the promoter sequence (Myanohara et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 1).
In addition, synthetic promoters that are not found in nature, can also function as a yeast promoters. For example, the sequence UAS one yeast promoter can be connected with a plot of activation of transcription of another yeast promoter, resulting in a synthetic hybrid promoter. Examples of such killed innych promoters are regulatory sequence of a gene ADH, combined with a plot of activation of transcription of the gene GAP (U.S. patent No. 4876197 and 4880734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of the gene composition ADH2, GAL4, GAL10, or RNA, combined with areas of activation of transcription of genes encoding glycolytic enzymes such as GAP genes or Hands (European patent application EP-A-0164556). Next, a yeast promoter may include naturally occurring promoters of non-yeast origin, characterized by the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, the elements described in Cohen et al., 1980, Proc. Natl. Acad. Sci. USA, 77, 1078; Henikoff et al., 1981, Nature, 283, 835; Hollenberg et al., 1981, Curr. Topics Environ. Immunol., 96, 119; Hollenberg et al., 1979, "The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae", In Plasmids of Medical, Environmental and Commercial Importance", eds. K.N.Timmis & A.Puhler; Mercerau-Puigalon et al., 1980, Gene, 11, 163; Panthier et al., 1980, Curr. Genet., 2, 109.
The DNA molecule can be expressed within cells of the yeast. The promoter sequence may be directly attached to the DNA molecule: in this case the first amino acid from the N-Terminus of the recombinant protein should always be a methionine, which is encoded by the start-codon ATG. If desirable, the methionine from the N-Terminus of the protein can be derived in vitro by incubation with cyanogenmod the om.
Chimeric proteins represent an alternative approach for yeast expressing systems to the same extent as for gene-expression systems, mammalian cells, baculoviruses and bacteria. Typically, the DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is drained from the 5'end of the heterologous coding sequence. After the expression of this construction will result in the formation of chimeras (hybrid) of two amino acid sequences. For example, the genes of the yeast or human superoxide dismutase (SOD) can be connected to the 5'-end of the foreign gene and subsequent expression in yeast. The DNA sequence at the junction of two amino acid sequences can encode the split site, and may not encode it: see, for example, European patent application EP-A-0196056. Another example is a chimeric obyedinenie protein. Such a chimeric protein construct so that preferably obyedinenie segment kept the site a target for enzyme processing (for example, specific processing ubicacin protease)causing cleavage ubicacin from a foreign protein. With this method, therefore, can be selected native alien proteins (see, for example, international patent application is a second application WO 88/024066).
On the other hand, foreign proteins can also be secreted from cells in a nutrient medium by creating chimeric DNA molecules that encode a chimeric protein comprising a leader sequence segment that provides secretion of the foreign protein from the yeast cells. Preferably should be coded processing sites located between the top segment and alien genome, which could be cleaved in vivo or in vitro. Leader segment typically encodes a signal peptide comprised of hydrophobic amino acids, which ensures secretion of this protein from the cell.
DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as invertase gene of yeast (European patent application EP-A-0012873; JPO 62096086) gene and the A-factor (U.S. patent No. 4588684). On the other hand, there are leader segments of non-yeast origin, such as the leader segment of the gene of interferon, which also provide for secretion in yeast (European patent application EP-A-0060057).
A preferred class providing the secretion leader segment are those leaders that use fragments of the gene αfactor, in which there is "pre" signal sequence, "Pro" Sigma is so Such variants of fragments αfactor, which can be used are full-size prepolymer αfactor (about 83 amino acid residue), as well as shortened versions of the signal segments αfactor (typically from about 25 to about 50 amino acid residues) (U.S. patent No. 4546083 and 4870008; European patent application EP-A-0324274). Additional leader segments, based on the leader α-factors that provide for secretion are hybrid α-factor leader segments collected in such a way that proposedvalue happen from the first yeast αfactor, and proposedvalue from the second yeast αfactor (see, for example, international patent application WO 89/02463).
Usually the sequence termination of transcription detected by yeast, presents regulatory segments, arranged in 3'-end with respect to the translational stop codon, i.e. they along with the promoter flank the coding sequence. These sequences provide the transcription of mRNA, which can then be translated into the polypeptide encoded by this DNA. Examples of the sequence termination of transcription and other recognizable by yeast termination sequences are those items that are included in SOS the AB genes encoding glycolytic enzymes.
Usually the above-described components, including the promoter, leader (if desired), are interested in the coding sequence and the sequence termination of transcription, contribute to each other in expressing the design. Expressing designs are often supported in the form of a replicon, such as an extrachromosomal element (e.g., a plasmid)that can stably exist in the body of the host, such as yeast or bacteria. This replicon may have two replication systems, this will maintain, for example, in yeast for the purpose of expression in prokaryotic host with the aim of cloning and amplification. Examples of such bifunctional bacterial-yeast vectors are YEp24 (Botstein et al., 1979, Gene 8, 17-24), pCl/1 (Brake et al., 1984, Proc. Natl. Acad. Sci. USA, 81, 4642-4646) and YRp17 (Stinchcomb et al., 1982, J. Mol. Biol., 158, 157). In addition, the replicon can be either vysokonapornoj or miscompiles the plasmid. Vysokogorya plasmid, in principle, be characterized by the presence of a number of copies from about 5 to about 200, and usually from about 10 to about 150. A host cell carrying vysokomobilnuju plasmid, preferably should bear at least about 10 and more preferably at least about 20 copies. The presence vysokonapornogo or discoloring vector can be from electrovan on the basis of influence, which has a vector and alien protein on the body of the host (see, for example, Brake et al., CIT. above).
On the other hand, expressing constructs can be integrated into the yeast genome using an integrating vector. Usually integrating vectors include at least one sequence homologous to the site of the yeast chromosome, which provides integration of this vector, and preferably includes two homologous sequences flanking expressing design. Presumably integration is a consequence of recombination between homologous DNA in the vector and the yeast chromosome (Orr-Weaver et al., 1983, Meth. Enzymol., 101, 228-245). Integrating vector may be directed to a specific locus of yeast by selecting the appropriate homologous sequence, which is included in this vector (see Orr-Weaver et al., CIT. above). One or more expressing constructs can be integrated so that, possibly, will lead to a reduction of the output level of the recombinant protein (Rine et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 6750). Chromosomal sequence, which is included in this vector can be either in the form of a single segment vector, which determines the integration of the entire vector, or in the form of two segments that are homologous to the next segme there chromosomes and flank expressing the design vector, resulting in a stable integration only expressing constructs.
Usually extrachromosomal expressing and integrating design can carry selective markers, providing selection of yeast strains that have been transformed. Selective markers may be biosynthetic genes that will be expressed in the cells of the host yeast, such as ADE2, HIS4, LEU2, TRP1, and resistance genes ALG7 and G418, which provide resistance of yeast cells, respectively, to tunicamycin and G418. In addition, suitable selective marker may also give the yeast the ability to grow in the presence of toxic substances such as metal ions. For example, the presence of a gene CUP1 provides the yeast growth in the presence of copper ions (Butt et al., 1987, Environ. Rev., 51, 351).
On the other hand, some of the above components can be incorporated together with each other in the composition of transforming vectors. Transforming the vectors typically include a selective marker that is either supported in the form of a replicon, or converted into an integrating vector as described above.
Expressing and transforming the vectors (in the form either of extrachromosomal replicons, or integrating vectors were constructed for the transformation of many species of yeast. For example, expressyou the vectors were constructed, among other things, for the following types of yeast: Candida albicans (Kurtz et al., 1986, Mol. Cell. Biol., 6, 142), Candida maltosa (Kunze et al., 1985, J. Basic Environ., 25, 141), Hansenula polymorpha (Gleeson et al., 1986, J. Gen. Environ., 132, 3459; Roggenkamp et al., 1986, Mol. Gen. Genet., 202, 302), Kluyveromyces fragilis (Das et al., 1984, J. Bacteriol., 158, 1165), Kluyveromyces lactis (De Louvencourt et al., 1983, J. Bacteriol., 154, 737; Van den Berg et al., 1990, Bio/Technol., 8, 135), Pichia guillerimondii (Kunze et al., 1985, J. Basic Environ., 25, 141), Pichia pastoris (Gregg et al., 1985, Mol. Cell. Biol., 5, 3376; U.S. patent No. 4837148 and 4929555), Saccharomyces cerevisiae (Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA, 75, 1929; Ito et al., 1983, J. Bacteriol., 153, 163), Schizosaccharomyces pombe (Beach & Nurse, 1981, Nature, 300, 706) and Yarrowia lipolytica (Davidow et al., 1985, Curr. Genet., 10, 380-471; Gaillardin et al., 1985, Curr. Genet., 10, 49).
Methods of introducing exogenous DNA into yeast cells-the hosts are well known in science and usually involve the transformation or spheroplasts or intact yeast cells treated with alkali cations. Transformation procedures usually vary depending on the type of yeast transformation which is carried out: see, e.g., Kurtz et al., 1986, Mol. Cell. Biol., 6, 142; Kunze et al., 1985, J. Basic Environ., 25, 141 (Candida); Gleeson et al., 1986, J. Gen. Environ., 132, 3459; Roggenkamp et al., 1986, Mol. Gen. Genet., 202, 302 (Hansenula); Das et al., 1984, J. Bacteriol., 158, 1165; De Louvencourt et al., 1983, J. Bacteriol., 154, 1165; Van den Berg et al., 1990, Bio/Technol., 8, 135 (Kluyveromyces); Gregg et al., 1985, Mol. Cell. Biol., 5, 3376; Kunze et al., 1985, J. Basic Environ., 25, 141; U.S. patent No. 4837148 and 4929555 (Pichia); Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA, 75, 1929; Ito et al., 1983, J. Bacteriol., 153, 163 (Saccharomyces); Beach & Nurse, 1981, Nature, 300, 706 (Schizosaccharomyces); Davidow t al., 1985, Curr. Genet., 10, 39; Gaillardin et al., 1985, Curr. Genet., 10, 49 (Yarrowia).
In this text, the term "antibody" refers to a polypeptide or group of polypeptides comprising at least one antigen-binding site. The term "antigen-binding site" denotes the spatial structure, surface characteristics and the distribution of charge which complementary parameters epitope antigen: this provides the binding of the antibodies with the corresponding antigen. The term "antibody" includes, for example, antibodies of vertebrates, chimeric antibodies, hybrid antibodies, gumanitarnye antibodies, altered antibodies, monovalent antibodies, Fab proteins and mono-domain antibodies.
Antibodies are specific proteins of the present invention, used for affinity chromatography, immunological tests for the detection and identification of proteins Nasseri.
Antibodies are specific proteins of the present invention, both polyclonal and monoclonal, can be obtained using standard methods. In General, protein, firstly, is used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are the preferred objects to obtain polyclonal sera due to developed the WMD significant amount of blood serum, as well as the availability of anti-rabbit and anticosti antibodies. Immunization is usually carried out by mixing or emulsification of a specific protein in saline (physiological) solution, preferably with an adjuvant such as complete Freund adjuvant, followed by injecting the mixture or emulsion parenterally (usually by subcutaneous or intramuscular injection). Usually a sufficient dose is 50-200 mcg per injection. Immunization is usually busterud (i.e. repeat) after 2-6 weeks by one or more injections of the protein in a salt solution, preferably with the inclusion of incomplete Freund adjuvant. Also, antibodies can be obtained by alternative through immunization in vitro using known in the art methods, which from the point of view of the purposes of the present invention can be equivalent to methods of immunization in vivo. Polyclonal antisera obtained by sampling blood from immunogenic animals in glass or plastic container, followed by incubation of the blood at 25°C for 1 hour and then incubation at 4°C for 2-18 hours. The whey is then allocate by centrifugation (e.g. at 1000g for 10 minutes). In rabbits during one sampling may be taken 20-50 cubes of blood.
Monoclonal antibodies get the standard method of Kohler-Milstein (Kohler & Milstein, 1975, Nature, 256, 495-496) or its modifications. Typically, in accordance with the above-described immunized mice or rats. However, unlike blood withdrawal in animals with the aim of obtaining serum remove the spleen (and optionally several large lymph nodes) and dissolved to a single cell. If it is desired, the spleen cells can be subjected to screening (after removal of non-specific adhesion of cells) by applying a cell suspension on a tablet or in a separate tablet hole, covered with a protein antigen. B-lymphocytes expressing associated with membrane immunoglobulin specific for the test antigen will bind on the tablet so that the remainder of the suspension is not washed from the tablet. Then spend the merging of the resulting b-lymphocytes or all of the dissolved splenocytes with myeloma cells, resulting in the formation of hybridoma, which are then grown in selective medium (for example, in the environment of the HAT containing gipoksantin, aminopterin and thymidine). The resulting hybridoma sown in a limited dilution and tested for production of antibodies that specifically bind with used for immunization antigen (and which is not associated with foreign antigens). Selected hybridoma, secreting monoclonal the specific antibodies (mAb), then cultured either in vitro (e.g. in the fermenters for hollow fibers or glass containers for tissue culture)or in vivo (in the ascitic fluid of mice).
If desired, antibodies (both polyclonal and monoclonal) can be labeled using standard methods. Suitable labels include fluorophores, chromophores, radioactive atoms (in particular,32R125I), electron-dense reagents, enzymes, and ligands, for which a known binding partners. Enzymes are usually revealed by their activity. For example, horseradish peroxidase is usually revealed by its ability to transform 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantitatively measured on the spectrophotometer. The term "specific binding partners" refers to a protein capable of binding the molecule-ligand, showing high level of specificity, as for example in the case of antigen and specific against him monoclonal antibody. Other examples of specific binding partners are Biotin and avidin (or streptavidin), immunoglobulin-G and a-protein, as well as numerous pairs of receptors and their ligands are known in the art. It should be clear that the above description does not purport to classification of various markers with their breakdown into any classes, because the RL one and the same label can be used in different modes. For example, the isotope125I can serve as a radioactive label or as electron-dense agent. Horseradish peroxidase can be either an enzyme or an antigen for monoclonal antibodies. In addition, it is possible to achieve the desired effect is to combine the different labels. For example, mAb and avidin in equal need for tagging in connection with the present invention, therefore, you can mark mAb Biotin and detect its presence using avidin marked, in turn, isotope125I, or by using antibioticnevada monoclonal antibodies labeled with horseradish peroxidase. Other options and features will be apparent to experts in the art and the scope of the present invention are considered as equivalent.
The pharmaceutical composition
The pharmaceutical composition may include a polypeptide, antibody or nucleic acid according to the present invention. These pharmaceutical compositions should contain a therapeutically effective amount of the polypeptides, antibodies or polynucleotides claimed by the present invention.
The term "therapeutically effective amount" in this text refers to the amount of therapeutic agent intended to treat, relieve symptoms or prophylact the key for a specific disease or condition or symptoms detectable therapeutic or preventative effect. This effect may be detected, for example, on levels of chemical markers or antigens. Therapeutic effects include a decrease in physical symptoms, such as lowering body temperature. The precise effective amount for a specific individual will depend on body weight and health, nature and severity of the disease condition, and the types of medicines or combinations thereof, selected for treatment. Therefore, there is no need for the original precise determination of the actual amount. However, the effective amount for a particular situation can be determined by routine experiments, such an approach is in the area of competence of the doctor.
For the purposes of the present invention, the effective dose should be in the range of from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg design DNA in the application to a specific individual, which it will be put.
The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means a carrier that is used for injecting drugs, such as antibodies or polypeptide, genes or other drugs. This term refers to any pharmaceutical carrier, to the which by itself does not induce the production of antibodies, dangerous for the individual receiving the pharmaceutical composition, i.e. which can be entered without binding him toxicity. Suitable carriers may be large, slowly metabolisable macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactivated viral particles. Such carriers are well known to specialists in this field of technology.
Can be used pharmaceutically acceptable salts, for example salts of inorganic acids, such as hydrochloride, hydrobromide, phosphates, sulfates and the like, or organic acid salts such as acetates, propionate, malonate, benzoate and the like. A detailed discussion of pharmaceutically acceptable excipients is available in the directory Remington's Pharmaceutical Sciences (Mack Publ. Co., N.J, 1991).
Pharmaceutically acceptable carriers comprising therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. In addition, such compositions may be present auxiliary excipients such as wetting or emulsifying agents, buffer components used to regulate the pH, and the like. Usually, therapeutic composition is prepared in the form of compositions for injection in the form of either solutions or suspen is s; solid forms suitable for solution or suspension to form a liquid fillers before injection can also be prepared. Also in the definition of the pharmaceutically acceptable carrier is activated liposome.
After preparation of the pharmaceutical compositions of the present invention they can be entered directly to the patient. Patients whose treatment is carried out, can be animals; in particular, the treatment may be conducted in respect of the person.
Direct delivery of the compositions, in principle, be accomplished by injection, subcutaneous, intraperitoneal, intravenous or intramuscular, or by delivery in the interstitial region. Also the composition can be introduced through damage (wound). Other routes of administration include oral and intra-lungs, in the form of suppositories, and transdermal or percutaneous introduction (see, for example, international patent application WO 98/20734), with needles, "gene guns" or hyposprays. Dosage may include the introduction of single or multiple doses.
Vaccines in accordance with the present invention may either be prophylactic (i.e. to prevent infection)or therapeutic (i.e. to treat already existing disease).
Such vaccines include immunizes the th antigen (antigens), the immunogen (immunogen), polypeptide (a polypeptide), protein (protein) or nucleic acid, usually in combination with "pharmaceutically acceptable carriers", which are any carriers, which by themselves do not induce the production of antibodies, dangerous for the individual receiving the composition. Suitable carriers are typically large, slowly metabolisable macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregation (such as oil droplets or liposomes), and inactivated viral particles. Such carriers are well known to specialists in this field of technology. In addition, these carriers can function as Immunostimulants ("adjuvants"). Moreover, the antigen or immunogen may be connected to a bacterial toxoid, such as diphtheria toxoid, tetanus toxoid, cholera toxoid, toxoid .pylori and other pathogens.
Preferred adjuvants designed to enhance the effectiveness of specific compositions are thus not limited to, (1) aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate and the like; (2) water-oil emulsion preparations (together with other specific Immunostimulants, such as morelove peptides [see below], or components of bacterial cell walls, or without them), such as, for example, (a) the drug MF59™ (application WO 90/14837; Chapter 10 in the book "Vaccine design: the subunit and adjuvant approach", eds. Powell &Newman, Plenum Press, 1995), containing 5% squalene, 0.5% tween-80 and 0.5% Span 85 (optionally containing various amounts of MTP-PE [see below], although this is not necessary), prepared in the form of submicron particles using microgenerator, such as microdispersion model 110 (Microfluidics, Newton, MA); (b) drug SAF, containing 10% squalene, 0.4% tween-80, 5% of the blocked polymer L121, and threonyl-MDP (see below or microdispersions in submicron emulsion or mixed with obtaining emulsions with larger particles, and (C) adjuvant system Ribi™ (RAS) (Ribi Immunochem, Hamilton, MT)containing 2% squalene, 0.2% tween-80, and one or more components of bacterial cell walls, including monophosphorylated-A (MPL), dimycolate-trehalose (TDM) and the preparation of cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponine adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, MA), can be used, as well as formed on the basis of their particles, such as ISCOM (immunostimulating complexes); (4) complete Freund adjuvant (CFA) and incomplete Freund adjuvant (IFA); (5) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7,IL-12 and others), interferons (e.g., γ-interferon), colony stimulating factor, macrophage (M-CSF), tumor necrosis factor TNF and others; and (6) other substances and compounds that act as Immunostimulants, providing increased efficiency in the song. Preferred are the aluminum salts and the drug MF59™.
As mentioned above, morelove peptides include, thereby not limited to, N-acetylmuramyl-L-threonyl-D-isoglutamine (threonyl-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)-ethylamine (MTP-PE), etc.
Immunogenic compositions (e.g., immunity antigen, or immunogen, or polypeptide, or protein, or nucleic acid together with a pharmaceutically acceptable carrier and adjuvant) should usually contain solvents, such as water, saline, glycerol, ethanol, etc. in Addition, in such fillers may include auxiliary components, such as wetting or emulsifying components, buffer components used for pH control, and the like.
Usually immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; can also be prepared in solid form or in suspension in a liquid fillers nepo is directly before injection. Also, the drug can be emulsified or encapsulated in liposomes to enhance adjuvant effect, as discussed above when considering pharmaceutically acceptable carriers.
Immunogenic compositions used as vaccines contain immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other observed from the above components, if necessary. By "immunologically effective amount" refers to the introduction of such amount to the subject either as a single dose or series of doses, which results in an effective treatment or prevention. This number varies depending on the health and physical condition of the particular subject, whose treatment, the taxonomic group to which the subject belongs (for example, nonhuman apes, primates in General, and so on), the ability of the individual's immune system to synthesize antibodies, the desired level of protection, a vaccine composition, individual evaluation by the attending physician, and other related factors. It is assumed that the analyzed number must fall within a fairly wide range, which can be quantitatively determined using routine methods.
Immunogenic compositions normally are entered by injecting the uti, for example, by injection - subcutaneous, intramuscular or transdermal (through the skin) (for example, international patent application WO 98/20734). Additional drugs that are suitable for other routes of administration, are preparations for oral and intra-lungs injection, suppositories, and transdermal preparations. Dosage may be composed of single doses or multiple doses. The vaccine can be given along with other immunoregulatory agents.
Alternatively, the vaccine based on the protein component can be applied DNA vaccination (see, e.g., Robinson & Torres, 1997, Seminars in Immunol., 9, 271-283; Donnelly et al., 1997, Annu. Rev. Immunol., 15, 617-648; see later in this text).
Fixtures for gene delivery
Gene therapy device designed for delivery of constructs carrying the coding sequence of therapeutic value of the present invention, which is intended for delivery to a mammal, in the body which should be its expression, can be administered either locally or systemically. These designs can be based on viral or non-viral vector approaches implemented in the models in vivo or ex vivo. The expression of such coding sequences can be induced using endogenous promoters milk is itausa or heterologous promoters. The expression of the coding sequence in vivo can be both constitutive and inducible (adjustable) character.
In the present invention included a gene-delivery system that can Express the represented nucleotide sequence. This gene delivery system is preferably based on viral vector and, more preferably, retroviral, adenoviral, agenerating (AAV) viral, herpes and alphaviruses vector. The viral vector can also be astroviruses, corona-virus, orthomyxoviruses, papovaviruses, paramyxoviruses, parvovirus, picornavirus, poxvirus or togaviruses: as overviews, see Jolly, 1994, Cancer Gene Therapy, 1, 51-64; Kimura, 1994, Hum. Gene Therapy, 5, 845-852; Connelly, 1995, Hum. Gene Therapy, 6, 185-193; Kaplitt, 1994, Nature Genet., 6, 148-153.
Retroviral vectors are well known in the art, the applicants believe that any retroviral gene therapy vector is applicable in connection with the present invention, including retroviruses types b, C and D, xenotropic retroviruses (for example, strains NZB-X1, NZB-X2 and NZB9-1 [see O'neill, 1985, J. Virol., 53, 160]), polytropic retroviruses, such as MCF and MCF-MLV (see Kelly, 1983, J. Virol., 45, 291), spumaviruses and lentiviruses; see "RNA Tumor Viruses", 2d ed., Cold Spring Harbor Lab., 1985.
Fragments of retroviral gene therapy vector can occur from different retroviruses. In the example, included in the retroviral vector of the long terminal repeats (LTR) can occur from sarcoma virus of mice, a tRNA binding site, from rous sarcoma virus, "packaging signal" from leukosis virus of mice, and the site of the beginning of the replication of the second chain from leukosis virus of birds.
These recombinant retroviral vectors can be used to create competent by transduction with retroviral vector particles, which is achieved by depositing them into an appropriate "packaging cell line" (see U.S. patent No. 5591624). Retroviral vectors can be constructed to ensure site-specific integration within the DNA of the host cell that can be achieved by introducing chimeric integranova enzyme in the composition of the viral particle (see international patent application WO 96/37626). Preferably, such a recombinant viral vector was a recombinant virus that is replication defective.
"Packaging cell line", suitable for use for the above-described retroviral vectors, are well known in the art, they can be easily created (see international patent application WO 95/30763 and WO 92/05266) and can be used to create a production cell lines (also called "vector cell lines", or "VCL"), is required for the production of recombinant vector of the s particles. Preferably, the packaging cell line are formed from the parent human cells (e.g., cell line NT) or parental cell lines mink, which excluded inactivation of human serum.
The preferred retroviruses suitable for construction of retroviral gene therapy vectors are leukosis virus of birds, leukosis virus bovine leukemia virus of mice, virus induction of foci in cells mink, sarcoma virus, mouse, virus reticuloendotheliosis and rous sarcoma virus. Specifically preferred viruses leukemia mice strains are A and A (Hartley & Rowe, 1976, J. Virol., 19, 19-25), sarcoma virus of Abelson ATSS No. VR-999), sarcoma virus Freund (ATSS No. VR-245), sarcoma viruses, Graffi and gross (ATSS No. VR-590), viruses of Kirsten sarcoma, Harvey and Rausher ATSS No. VR-998) and leukosis virus of mice, Malone ATSS No. VR-190). These retroviruses can be obtained from depositories or collections such as the American type culture collection ("ATSS"), located in Rockville (Maryland), or isolated from known sources using standard techniques.
Examples of known retroviral gene therapy vectors that are applicable in the scope of the present invention, are those vectors, which are described in patent applications - British GB 2200651, European EP 0415731, EP 0345242, EP 0334301 international WO 89/02468, WO 89/05349, WO 89/09271, WO 90/02806, WO 90/07936, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, WO 91/02805, WO 91/02825, WO 95/07994, U.S. patents№№ 5219740, 4405712, 4861719, 4980289, 4777127, 5591624; see also Vile, 1993, Cancer Res., 53, 3860-3864; Vile, 1993, Cancer Res., 53, 962-967; Ram, 1993, Cancer Res., 53, 83-88; Takamiya, 1992, J. Neurosci. Res., 33, 493-503; Baba, 1993, J. of Neurosurgery, 79, 729-735; Mann, 1983, Cell, 33, 153; Cane, 1984, Proc. Natl. Acad. Sci. USA, 81, 6349; Miller, 1990, Hum. Gene Therapy, 1.
Viruses for gene therapy based on the human adenoviruses are also well known in science and applicable in connection with the present invention: see, for example, Berkner, 1988, Biotechniques, 6, 616 and Rosenfeld, 1991, Science, 252, 431, and international patent application WO 93/07283, WO 93/06223 and WO 93/07282. Examples of known adenoviral gene therapy vectors that are applicable for the purposes of the present invention, are those vectors that are described in the sources cited above and in international patent applications WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984, WO 95/00655, WO 95/27071, WO 95/29993, WO 95/34671, WO 96/05320, WO 94/08026, WO 94/11506, WO 93/06223, WO 94/24299, WO 95/14102, WO 95/24297, WO 95/02697, WO 94/28152, WO 94/24299, WO 95/09241, WO 95/25807, WO 95/05835, WO 94/18922 and WO 95/09654. On the other hand, can be applied to the introduction of DNA connected to killed adenovirus described in Curiel, 1992, Hum. Gene Therapy, 3, 147-154. Gene-delivery system of the present invention can also be based on adeno-associated viruses (AAV). Preferred examples of such vectors in connection with their use on this image is the shadow are vectors, based on the strain AAV-2, as claimed by Srivastava (Srivastava) in international patent application WO 93/09239. The most preferred AAV-vectors includes two inverted terminal repeat of AAV genome, in which the native D-sequence modified by nucleotide substitutions so that at least five native nucleotides and up to 18 native nucleotides, and preferably at least 10 native nucleotides and up to 18 native nucleotides, and most preferably 10 native nucleotides are retained, and the remaining nucleotides of the composition of the D-sequence deleteroute or replace negativnye nucleotides. Native D-sequences from the structure of the inverted terminal repeats of AAV are 20-nucleotide motifs, available in each terminal terminal repeat of AAV (i.e. there is one such motif in each end section)that do not participate in education HP. Negativly replacement nucleotide can be any nucleotide, in addition to the nucleotide present in the composition of natural D-sequence in the same position. Other applicable examples of vectors based on AAV viruses are strains pWP-19 and pWN-1, both of these are described in Nahreini, 1993, Gene, 124, 257-262. Another example of such AAV-vector is a vector of psub201 (see Samulski, 1987, J. Virol., 61, 3096). Another example of an AAV-vector is a vector of Double-D ITR. To strairway vector Double-D ITR described in U.S. patent No. 5478745. Other vectors are vectors, Carter declared in U.S. patent No. 4797368 and Muzyczka in U.S. patent No. 5139941 and Chartejee in U.S. patent No. 5474935 and Kotin in the international application WO 94/288157. Another example of AAV vectors, applicable in connection with the present invention is to design SSV9AFABTKneo, it includes the AFP enhancer and the promoter of the gene of albumin and provides the expression predominantly in the liver. Its structure and design are described in Su, 1996, Hum. Gene Therapy, 7, 463-470. Another series of AAV vectors for gene therapy are described in U.S. patent No. 5354678, 5173414, 5139941 and 5252479.
Vectors for gene therapy in accordance with the present invention include vectors based on herpesvirus. The most acceptable and preferred examples are vectors based on simple herpesvirus comprising a sequence that encodes a polypeptide timedancing, such as those vectors, which are described in U.S. patent No. 5288641 and European application EP 0176170 (Roizman). Additional examples of vectors based on simple herpesvirus are HFEM/ICP6-LacZ disclosed in WO 95/04139 (Wistar Institute), pHSVlac described in Geller, 1988, Science, 241, 1667-1669 and in international patent your WO 90/09441 and WO 92/07945; vector HSV Us3::pgC-lacZ, described by Fink, 1992, Hum. Gene Therapy, 3, 11-19, and vector HSV 7134, 2-RH-105 and GAL4 described in European application EP 0453242 (Breakefield), as well as those included in the collection of ADS Depository under No. the TCC VR-977 and ATSS VR-260.
Also represented gene therapy vectors based on the alpha viruses, which can be used in connection with the present invention. Preferred are vectors based alphavirus Sindbis. Togavirus, virus, Semliki Forest (ATSS VR-67; ATS VR-1247), the virus of Middelburg ATS VR-370), the virus Ross river (ATSS VR-373; ATS VR-1246), the virus Venezuelan equine encephalitis (ATSS VR-923; ATS VR-1250; ATS VR-1249; ATS VR-532), and those togavirus, which are described in U.S. patent No. 5091309 and 5217879 and in international patent application WO 92/10578. More specifically, applicable alphavirus vectors, which are described in application for U.S. patent No. 08/405627 dated March 15, 1995, in international patent applications WO 94/21792, WO 92/10578 and WO 95/07994 and in U.S. patent No. 5217879 and 5091309. Such alpha viruses may be available from depositories or collections such as the collection of ATS, located in Rockville (Maryland), or isolated from known sources using standard methods. Preferably should be used alphavirus vectors, characterized by low cytotoxicity (see USSN No. 08/679640).
DNA vector systems such as eukaryotic expression systems can also be used for expression of the nucleic acids of the present invention: see international patent application WO 95/07994, which has a detailed description of eukaryotic ek is pressione systems. Preferably eukaryotic expression system of the present invention is based on alphavirus vectors, and most preferably in the vector, which is the genome of Sindbis virus.
Other viral vectors suitable for use from the point of view of the practice of the present invention, include those vectors that are derived from poliovirus, for example ATSS VR-58 and those described by Evans, 1989, Nature, 339, 385, and Sabin, 1973, J. Biol. Standart., 1, 115; from rhinoviruses, for example ATSS VR-1110 and those described in Arnold, 1990, J. Cell. Biochem., L401; poxviruses, such as the Canary poxvirus and vaccinia virus, for example ATCC VR-111 and ATSS VR-2010 and those described by Fisher-Hoch, 1989, Proc. Natl. Acad. Sci. USA, 86, 317; Flexner, 1989, Ann. N.Y. Acad. Sci., 569, 86; Flexner, 1990, Vaccine, 8, 17; U.S. patent No. 4603112 and 4769330 and international patent application WO 89/01973; from monkey virus, SV40, for example ATSS VR-305 and those described in Mulligan, 1979, Nature, 277, 108 and Madzak, 1992, J. Gen. Virol., 73, 1533; influenza virus, for example ATSS VR-797 and recombinant influenza viruses used in the methods of reverse genetics in accordance with the description of U.S. patent No. 5166057 and Enami, 1990, Proc. Natl. Acad. Sci. USA, 87, 3802-3805; Enami & Palese, 1991, J. Virol., 65, 2711-2713 and Luytjes, 1989, Cell, 59, 110 (see also McMichael, 1983, New England J. Med., 309, 13 and Yap, 1978, Nature, 273, 238 and Nature, 1979, 277, 108); from human immunodeficiency virus as described in EP 0386882 and Buchshacher, 1992, J. Virol., 66, 2731; from measles virus, for example ATSS VR-67 and ATSS VR-1247 and those described in EP 0440219; from auriferous, for example ATSS VR-368; from Bebaru virus, such as ATSS VR-600 and ATSS VR-1240; from Cabassou virus, such as ATSS VR-922; from Chikungunya virus, such as ATSS VR-64 and ATSS VR-1241; from virus, Fort Morgan, for example ATSS VR-924; from Getah virus, such as ATSS VR-369 and ATSS VR-1243; from gyzylagachskom virus, such as ATSS VR-927; from Mayaro virus, such as ATSS VR-66; from Mucambo virus, such as ATSS VR-580 and ATSS VR-1244; from Ndumu virus, such as ATSS VR-371; from Pixuna virus, such as ATSS VR-372 and ATSS VR-1245; from Tonate virus, such as ATSS VR-925; from Triniti virus, such as ATSS VR-469; from Una virus, such as ATSS VR-374; from Whataroa virus, such as the VR ADS-926; virus Y-62-33, for example ATSS VR-375; from virus Nyong O (virus, Eastern encephalitis), for example ATSS VR-65 and ATSS VR-1242; virus Western encephalitis, for example ATSS VR-70, ATSS VR-1251, ATSS VR-622 and ATSS VR-1252; and coronaviruses, such as ATSS VR-740 and those described in Hamre, 1966, Proc. Soc. Exp. Biol. Med., 121, 190.
The delivery compositions of the present invention into cells is not limited to the use described above of viral vectors. Can also be applied to other delivery methods and environments, such as, for example, nucleotide expressing vectors with polycation Packed DNA, connected or not connected to killed adenovirus (see, for example, an application for a U.S. patent is the 08/366787, filed December 30, 1994, and Curiel, 1992, Hum. Gene Therapy, 3, 147-154; DNA, coupled with the ligand: see, for example, Wu, 1989, J. Biol. Chem., 264, 16985-16987; delivery system cells to eukaryotic cells: see, for example, patent application U.S. No. 08/240030, filed may 9, 1994, and patent application U.S. No. 08/404796; depositing setprimarykey hydrogel material; the use of "manual of gene gun" as described in U.S. patent No. 5149655; the use of ionizing radiation as described in U.S. patent No. 5206152 and international patent application WO 92/11033; the use of "the charge neutralization nucleotide or merge with the cell membrane. In addition, such approaches are described in Philip, 1994, Mol. Cell. Biol., 14, 2411-2418 and Woffendin, 1994, Proc. Natl. Acad. Sci. USA, 91, 1581-1585.
Can be applied a method of gene delivery particles (see, for example, an application for U.S. patent No. 60/023867. Briefly, the sequence can be embedded in the standard vectors, which have the usual regulatory sequence that provides high-intensity expression; then carry out incubation with synthetic gene-delivery molecule, such as polymeric DNA-binding cations, such as polylysine, Protamine, and albumin, linked with ligands, marking cells, such as Belorusskoe, as described in Wu &Wu, 1987, J. Biol. Chem., 262, 4429-442, or insulin as described in Hucked, 1990, Biochem. Pharmacol., 40, 253-263, or galactose as described in Plank, 1992, Bioconjugate Chem., 3, 533-539, or lactose, or transferrin.
Can also be used open ("naked") DNA. Examples of methods of making an open-DNA are the techniques that are described in international patent application WO 90/11092 and U.S. patent No. 5580859. The absorption efficiency can be increased with the use of balls made of latex, which are destroyed in the biological environment. DNA loaded in the balls of latex, efficiently delivered into cells in the endocytosis, which is triggered by these balls. This method can be improved by treating the beads with the aim of increasing hydrophobicity, thereby facilitates the destruction of endosome and the output of this DNA into the cytoplasm.
Liposomes that can be used as gene delivery systems described in U.S. patent No. 5422120, in international patent applications WO 95/13796, WO 94/23697 and WO 91/14445 and EP 524968. As described in U.S. patent application No. 60/023867 non-viral delivery method, the nucleotide sequence encoding the polypeptide, can be integrated into standard vectors, which include the usual regulatory sequence that provides intense expression, followed by incubation with synthetic g is n-delivery molecule, such as polymeric DNA-binding cations, such as polylysine, Protamine, and albumin, linked with ligands, marking cells, such as Belorusskoe, insulin, galactose, lactose, or transferrin. In other delivery systems, liposomes are used to encapsulate DNA, the carrier of a particular gene under the control of tissue-specific or origitano active (i.e. not dependent on the tissue type) promoters. Other non-viral delivery systems are mechanical delivery systems such as those used in the method described in Woffendin et al., 1994, Proc. Natl. Acad. Sci. USA, 91, (24), 11581-11585. Moreover, the coding sequence and its expression product can be delivered by depositing setprimarykey hydrogel materials. Can be used with other standard methods of gene delivery, including coding sequences, for example, which are based on the use of "manual of gene gun" as described in U.S. patent No. 5149655, on the application of ionizing radiation, providing activation of transferred genes, as described in U.S. patent No. 5206152 and international patent application WO 92/11033.
Examples and liposomal poly-gene-delivery systems are those systems described in U.S. patent No. 5422120 and 4762915, international PA is entih applications WO 95/13796, WO 94/23697 and WO 91/14445, in EP 0524968 and Stryer, 1975, "Biochemistry", ed. W.H.Freeman, San Francisco, pp. 236-240; Szoka, 1980, Biochem. Biophys. Acta, 600, 1; Bayer, 1979, Biochem. Biophys. Acta, 550, 464; Rivnay, 1987, Meth. Enzymol., 149, 119; Wang, 1987, Proc. Natl. Acad. Sci. USA, 84, 7851; Plant, 1989, Anal. Biochem., 176, 420.
Polynucleotide composition may include a therapeutically effective amount of gene therapy system in accordance with the term specified above. For the purposes of the present invention, the effective dose should be in the range of from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg DNA constructs in application to the individual, which they are entered.
After inclusion in a pharmaceutical composition such polynucleotide compositions of the present invention can be (1) directly injected into a patient; (2) delivered ex vivo to cells derived from this patient; or (3) used for expression of recombinant proteins in vitro. The subjects, which will be introduced such compositions may be mammals or birds. The treatment may be carried out in relation to the people.
Direct introduction of the compositions, in principle, should be carried out by injection - subcutaneous, intraperitoneal, intravenous or intramuscular, or they must be entered in the interstitial space. Also these compositions can be introduced into damage (wounds). Other routes of administration are what I oral and intra-lungs introduction, the use of suppositories, and transdermal introduction or introduction "through the skin" (see, for example, international patent application WO 98/20734), or introduction to the use of needles, "gene guns" or hyposprays. The dosage can be composed of single or multiple doses.
Ways of introducing ex vivo with subsequent re-implantation of the transformed cells to a subject are well known in the art and described, for example, in international patent application WO 93/14778. Examples of cells suitable for introduction in the embodiment, ex vivo, are, for example, stem cells, in particular hematopoietic cells, lymphatic cells, macrophages, dendritic cells or tumor cells.
In General, delivery of nucleic acid variants in the application of ex vivo and in vitro can be carried out using, for example, such procedures: transfection mediated by dextran, precipitation with calcium phosphate, transfection with polybrene, the fusion of protoplasts, electroporation, encapsulation of polynucleotide (polynucleotides) in liposomes, and direct microinjection DNA in the nucleus, all of these methods are well known in science.
Pharmaceutical compositions containing polynucleotide andpolypeptides
In addition to the pharmaceutically acceptable carriers and salts, which have been described above, can be used the following aircraft is magadelene components, to be included in the compositions containing polynucleotide and / or polypeptide.
One example are polypeptides, which are thus not limited to, include Belorusskoe (ASOR); transferrin; asialoglycoprotein; antibodies; antibody fragments; ferritin; interleukins; interferons; colony-stimulating factor for granulocytes and macrophages (GM-CSF), colony stimulating factor, granulocyte (G-CSF), colony stimulating factor, macrophage (M-CSF), stem cell factor, and erythropoietin. Can also be used viral antigens such as envelope proteins. Can also be used for other proteins of pathogenic organisms, such as 17-amino acid peptide malaria parasite stage round sporozoite known as RII.
B. Hormones, vitamins, etc.
Other groups of compounds that can be included in the composition include, for example, hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, such as folic acid.
C. Polyalkylene, polysaccharides, etc.
Also polyalkyleneglycol can be included together with the desired polynucleotide or polypeptides. In a preferred embodiment, polyalkyleneglycol is polyethylene glycol. In addition, the composition can be included mono-, di - or polysaccharides. In the preferred embodiment of this aspect of gender is a saccharide is dextran or DEAE-dextran. Can also be used chitosan and poly(lactide-coglycolide).
D. Lipids and liposomes
Desirable polynucleotide or polypeptides can be encapsulated in a lipid or packaged in liposomes prior to delivery to a subject or derivatives of the cell.
Lipid encapsulation is usually performed with the use of liposomes, which can stably bind or turn inward nucleic acid. The ratio of Packed polynucleotide and lipid material can vary, but generally close to 1:1 (mg of DNA for μmol lipid), or the number of lipid slightly higher. The use of liposomes for delivery of nucleic acids as an overview, see Hug & Sleight, 1991, Biochim. Biophys. Acta, 1097, 1-17; Straubinger, 1983, Meth. Enzymol., 101, 512-527.
Liposomal preparations for use in the present invention include cationic (i.e. positively charged), anionic (i.e. negatively charged) and neutral preparations. Cationic liposomes have been shown to provide intracellular delivery of plasmid DNA (Felgner, 1987, Proc. Natl. Acad. Sci. USA, 84, 7413-7416), mRNA (Malone, 1989, Proc. Natl. Acad. Sci. USA, 86, 6077-6081) and purified transcription factors (Debs, 1990, J. Biol. Chem., 265, 10189-10192), preserving its functionality.
Cationic liposomes are readily available. For example, N-[(1,2,3-dialerace)propyl]-N,N,N-triethylammonium (DOTMA) liposomes is available under the trademark "Lipofectin" from the company Gibco BRL (Grand Island, NY) (see also Felgner, CIT. above). Other available on a commercial basis liposomes are transfected (DDAB/DOPE) and DOTAP/DOPE (Boehringer). Other cationic liposomes can be prepared from readily available materials using methods well known in the art: see, for example, Szoka, 1978, Proc. Natl. Acad. Sci. USA, 75, 4194-4198; and the synthesis of DOTAP (1,2-bis(oleolux)-3-(ammonium)propane) liposomes are described in international patent application WO 90/11092.
Similarly readily available are anionic and neutral liposomes, such as obtained from the company Avanti Polar Lipids (Birmingham, AL), or can be easily obtained using readily available materials. Such materials include, without limitation, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPG), dioleoylphosphatidylcholine (DOPE). These materials can also be mixed with the source material DOTMA and DOTAP in appropriate ways. The methods of preparation of liposomes with the use of these materials is well known in science.
Liposomes can include a multilayer vesicles (MLV), small single-layer vesicles (SUV) or large single-layer vesicles (LUV). Various complexes of liposomes and nucleic acids form using methods well known in the art: see, for example, Straubinger, 1983, Meth. Immunol., 01, 512-527; Szoka, 1978, Proc. Natl. Acad. Sci. USA, 75, 4194-4198; Papahadjopoulos, 1975, Biochim. Biophys. Acta, 394, 483; Wilson, 1979, Cell, 17, 77; Deamer & Bangham, 1976, Biochim. Biophys. Acta, 443, 629; Ostro, 1977, Biochem. Biophys. Res. Commun., 76, 836; Fraley, 1979, Proc. Natl. Acad. Sci. USA, 76, 3348; Enoch & Strittmatter, 1979, Proc. Natl. Acad. Sci. USA, 76, 145; Fraley, 1980, J. Biol. Chem., 255, 10431; Szoka &Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. USA, 75, 145; Schaefer-Ridder, 1982, Science, 215, 166.
In addition, lipoproteins can be combined with polynucleotide or polypeptide intended for delivery. Examples of lipoproteins, which can be used are: chylomicrons, HDL, IDL, LDL and VLDL. Can also be used mutants, fragments and chimeras of these proteins. Can also be used modifications of naturally occurring lipoproteins, such as acetylated LDL. These lipoproteins can deliver polynucleotides to specific cells expressing receptors for lipoproteins. Preferably, in the case of the inclusion in the composition of lipoproteins along with intended for delivery to polynucleotides other ligands, providing targeted delivery of the targets were not included in the composition of such a composition.
Naturally occurring lipoproteins are composed of lipid and protein components. The protein component known as the "apoprotein". Currently, were isolated and identified apoprotein a, b, C, D and E. at least two who C these classes are available at several proteins, which are indicated by Roman numerals: AI, AII, AIV; CI, CII, CIII.
In lipoprotein composition may contain more than one apoprotein. For example, naturally occurring chylomicrons include apoprotein a, b, C and E, and over time they lose apoprotein and acquire apoprotein C and E. the composition of VLDL includes apoprotein a, b, C, and e in the composition of the LDL - apoprotein In, and in the composition of HDL include apoprotein a, C and E.
The amino acid composition of these apoproteins known and described, for example, Breslow, 1985, Annu. Rev. Biochem., 54, 699; Law, 1986, Adv. Exp. Med. Biol., 151, 162; Chen, 1986, J. Biol. Chem., 261, 12918; Kane, 1980, Proc. Natl. Acad. Sci. USA, 77, 2465; Utermann, 1984, Hum. Genet., 65, 232.
The composition of lipoproteins consist of a variety of lipids, including triglycerides, cholesterol (free or in the form of esters and phospholipids. The lipid composition varies in naturally occurring lipoproteins. For example, the chylomicrons are mainly triglycerides. A more detailed description of the lipid composition of naturally occurring lipoproteins can be found, for example, 128-m log volume "Methods in Enzymology" (1986). The lipid composition is chosen in such a way as to correspond to the conformation of apoprotein, thereby providing activity by binding to the appropriate receptor. The lipid composition can also be chosen so as to facilitate the hydrophobic interaction and the binding is compared with the molecule, linking polynucleotide.
Naturally occurring lipoproteins may, for example, be isolated from serum by ultracentrifugation. Some of the methods described in "Methods in Enzymology (CIT. above) and Pitas, 1980, J. Biochem., 255, 5454-5460 and Mahey, 1979, J. Clin. Invest., 64, 743-750. Lipoproteins can also be obtained in vitro or by using recombinant technology by gene expression apoproteins in the desirable cell-host: see, for example, Atkinson, 1986, Annu. Rev. Biophys. Chem., 15, 403, and Radding, 1958, Biochim. Biophys. Acta, 30, 443. The lipoproteins can be purchased from commercial distributors such as Biomedical Technologies Inc. (Stoughton, MA, USA). Additional description of lipoproteins can be found at Zuckermann et al. in the application PCT/US 97/14465.
In the composition comprising the desired polynucleotide or polypeptide whose delivery is expected, can be included poly agents, lipoprotein or without it.
Usually poly-components are characterized by the total positive charge at physiological pH values and is able to neutralize the electrical charge of nucleic acids to facilitate delivery to the desired location. These components can be used both in vitro and ex vivo and in vivo. Poly-components can be used for delivery of nucleic acids in vivo p is the patients by intramuscular, subcutaneous injection, etc.
These polypeptides are examples of applicable poly component: polylysin, polyalanine, poliorcetes and Protamine. Other examples are the histones, Protamine, human serum albumin, DNA-binding proteins, nonhistone chromosomal proteins, capsid proteins, DNA viruses such as virus H; transcription factors also include the sites of DNA binding, and therefore they can also be used as agents, condensing the nucleic acid. Basically, the core domain, which binds DNA, is part of such transcription factors as WITH/SEVRES, c-jun, c-fos, AP-1, AR-2, AR-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP and TFIID.
Organic poly-components are spermine, spermidine and putrescine.
Dimensions and physical properties of poly-component can be extrapolated on the basis of the above list, with the aim of constructing other poly-polypeptide components of nature or to create synthetic poly components.
Synthetic poly-components, which are applicable include, for example, DEAE-dextran and polybrene. Lipofectin™ and lipofectamine™ are the monomers that form the poly-complexes when combined with polynucleotide/poly is eptide.
Immunological diagnostic tests
Antigens Nasseri of the present invention can be used in immunological tests, aimed at determining the levels of antibodies (or, on the other hand, antibodies to antigens of Neisseria can be used to determine the levels of antigens). Immunological tests based on well-defined recombinant antigens can be developed to replace invasive diagnostic tests. Antibodies to proteins of Neisseria can be detected in the composition of biological samples, such as blood or serum. The development of such immunological tests is subject to numerous modifications and their wide range is known in the art. Recipe of these immunological tests can be based, for example, in tests with competitive binding, or direct reaction, or the "sandwich"principle. In these protocols, for example, can be used with a solid substrate or immunoprecipitate. Many tests are labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive molecules or dye molecules. Also known tests that use the amplification of the signals on the basis of the corresponding probe, the examples in this case I have are tests, using Biotin and avidin, and enzyme-labeled and mediated immunological tests, such as TYPHOID (enzyme-linked immunosorbent assay).
The reagent kits, applicable for immunodiagnostics, including appropriate labeled reagents, is formed by packaging the appropriate materials, including composition of the present invention, in suitable containers, along with other materials and reagents (e.g., appropriate buffers, salt solutions and the like)necessary for the conduct of a competitive test, as well as the necessary set of descriptive statements.
Hybridization of nucleic acids
The term "hybridization" refers to the linking of two nucleotide sequences to each other by hydrogen bonds. Usually one of these sequences is fixed on a solid substrate, and the other is in solution in a free state. Then provide the contact between the two sequences in such conditions that favor the formation of hydrogen bonds. Factors that affect the formation of such ties are the type and amount of solvent; reaction temperature; the duration of hybridization; mixing; the presence of components that would prevent the nonspecific binding in the liquid phase in which sledovatelnot solid substrate (denhardt's solution or reagent BLOTTO); the concentration of sequences; the use of compounds that can increase the speed of binding sequences (textresult or polyethylene glycol); and the rigidity of the washing conditions after hybridization (see Sambrook et al., CIT. above, volume 2, Chapter 9, pp. 9.47-9.57).
Under the "rigidity" refers to conditions for hybridization, which favor binding of very similar sequences in contrast to different sequences. For example, the combination of temperature and salt concentrations must be chosen so that it was at 120-200°C below the design temperature of dissociation (Tm) analyzed hybrid. The parameters of temperature and salt concentrations can be usually determined empirically in preliminary experiments in which samples of genomic DNA immobilized on the filters, hybridized with the analyzed sequence and then washed under conditions of varying degrees of stringency (see Sambrook et al., p. 9.50).
Variables that you must consider when implementing, for example, southern blotting, are (1) the complexity of the structure of DNA, which will be subjected to blotting, and (2) the level of homology of the probe and detected sequences. The total number of the investigated fragment (fragments) may vary on the order of 0.1 to 1 µg for plasma is s or phage or from 10 -9up to 10-8g for adenocarinoma gene composition of complex eukaryotic genome. For less complex polynucleotides can be used for significantly shorter periods of time blotting, hybridization and exposure, a smaller number of source polynucleotides and lower specific activity probes. For example, odnokorennye yeast gene can be detected during the exposure time of only 1 hour when the number of initial yeast DNA 1 µg, the blot for 2 hours and hybridization for 4-8 hours with a probe having an activity of 100 million pulses/min of 1 µg. For adenocarinoma gene of the mammal conservative approach is to start with 10 μg DNA, blotting during the night and hybridization overnight in the presence of 10% doctranslate using a probe with a specific activity of more than 100 million pulses/min 1 µg, resulting in the exposure time will be approximately 24 hours.
Several factors can affect the melting temperature (Tm) hybrid DNA/DNA formed between the probe and analyzed by fragment, and, therefore, the appropriate conditions for hybridization and washing. In many cases, the probe is not 100% homologous to the given fragment. Other commonly considered variables are the length and the total content of GC pairs in hybridizes placentas is alnoth, as well as ionic strength and the content of formamide in the hybridization buffer. The impact of all these factors can be accounted for by a single equation:
Tm= 81 + 16,6·(log10Ci) + 0,4·(% GC pairs) - 0,6·(% formamide)-600/n - 1,5·(% errors),
where Ci is the salt concentration (monovalent ions), and n is the length of this hybrid, expressed in number of base pairs (the formula is slightly modified compared with published Meinkoth &Wahl, 1984, Anal. Biochem., 138, 267-284).
When designing schemes hybridization experiment in a standard way can be modified some of the factors influencing hybridisation of nucleic acids. The most simple from the point of view of bringing to an optimum temperature of hybridization and washing and concentration of salts used in washing. When increasing the temperature at which the reaction is carried out hybridization (i.e., the stiffness of the reaction), it becomes less likely that hybridization will occur between nucleotide chains that are non-homologous, resulting in reduced background noise. If radioactively labeled probe is not completely homologous immobilized fragment (a situation common in the analysis of gene families and experiments on interspecific hybridization), the hybridization temperature should be reduced, then the background noise will increase. Temperature is RA flushing similarly affects the intensity of the hybridization band and the background level. The rigidity of the conditions of leaching also increases at lower concentrations of salts.
In General, the standard hybridization temperatures in the presence of 50% formamide are 42° (C) to probe homologous to the fragment-target on 95-100%, 37°by homology at the level of 90-95% and 32°when the level of homology 85-90%. For lower levels of homology content of formamide should be reduced from the subsequent corresponding bringing the temperature based on the above equations. If the level of homology between the probe and the fragment-target is unknown, the most simple approach is to experiment initially with soft conditions of hybridization and washing. If after autoradiographical analysis are intense bands of nonspecific binding and the level of background noise, the filter can be washed at high stringency and tested again. If the time required for exposure, makes this approach impractical, several options rigidity of the hybridization and / or washing can be tested simultaneously ("in parallel").
Tests with nucleotide probes
In such methods, like PCR, analysis of branched DNA probes and blotting techniques based on the use of nucleotide probes in accordance with the present invention, it is possible to identify KD is To or mRNA. They say that the probe "hybridize" sequence of the present invention when it forms a duplex or double-stranded complex, which is stable enough to be detected.
The respective nucleotide probes should hybridizat and nucleotide sequences Nasseri of the present invention (including coding and noncoding chain). Although the specific amino acid sequence can encode many different nucleotide sequences, the native sequence Nasseri are preferred because they are really present in the cells of these organisms. mRNA is the coding sequence, so the probe should be complementary to the corresponding coding sequence; single-stranded cDNA complementary to mRNA, therefore, cDNA-probe should be complementary to non-coding sequence.
It is not necessary that the nucleotide sequence of the probe was identical to the sequence of Neisseria (or its complement), some level of variability in the sequence and length of the nucleotide sequence may cause increase the sensitivity of the test when the nucleotide probe can form a duplex with nucleotides target, which can be what Yavin. Also nucleotide probe may include additional nucleotides, which are designed to stabilize the formed duplex. Additional sequence Neisseria can also perform a supporting role labels are required to identify the formed duplex. For example, complementary nucleotide sequence may be attached to the 5'-end of the probe, while the rest of the sequence of this probe is complementary to the sequence of Neisseria. On the other hand, complementary nucleotides or extension sequence can be dispersed along the length of the probe taking into account the fact that the nucleotide sequence of this probe is characterised by significant levels of complementarity to the sequence of Neisseria, the result will be provided with their hybridization and the formation of their duplex, which can be detected.
The exact length and nucleotide sequence of the probe will depend on the conditions of hybridization, such as temperature, salt conditions, and the like. For example, for diagnostic applications, depending on the complexity of the analyzed nucleotide sequence of the nucleotide probe usually consists of at least 10-20 nucleotides, preferably from 15 to 25, and most preferably at least neucleotides, although it may be shorter. Short of seed, usually require lower temperatures for the formation of a sufficiently stable hybrid complexes with the matrix.
The probes can be formed using the procedures of synthesis, for example using trivinho method Matteucci et al. (Matteucci et al., 1981, J. Amer. Chem. Soc., 103, 3185), or in accordance with Urdea et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 7461, or made commercially available automated oligonucleotide synthesizers.
The chemical nature of the probe can be selected based on specific preferences. For some applications, the preferred are DNA or RNA. For other uses may be made of different modifications: for example, modification of the axial structure, such as phosphothioate or methylphosphonate, can be used to extend the half-life in vivo, changes in the level of affinity of RNA, increasing resistance to the action of nucleases and the like (see, for example, Agrawal & Iyer, 1995, Curr. Opinion Biotechnol., 6, 12-19; Agrawal, 1996, TIBTECH, 14, 376-387); can also be used such analogues as peptides nukleinovykh acids (see, for example, Corey, 1997, TIBTECH, 15, 224-229; Buchardt et al., 1993, TIBTECH, 11, 384-386).
On the other hand, polymerase chain reaction (PCR) is another well known way of detecting small amounts of nucleic acid targets. This test OPI is an by Mullis et al. (Mullis et al., 1987, Meth. Enzymol., 155, 335-350) and in U.S. patent No. 4683195 and 4683202. Two nucleotide"seed" hybridized with nucleic acid target and is used to seed the reaction. Such priming may include a sequence that is not hybridized sequence designed for amplification of the target (or its complement), in order to give stability to the duplex, or, for example, to make a standard restriction site. Typically, such a sequence must planirovati desired sequence of Neisseria.
Thermostable polymerase creates copies of a nucleotide acid targets from seed, using the original nucleic acid-target as a matrix. After this polymerase provides the appearance of a certain threshold number of nucleic acid targets, they can be detected with the use of more traditional methods such as southern blotting. When using the southern blot labeled probe should hybridizing sequence of Neisseria (or its complement).
Also the mRNA or cDNA can be identified through traditional blotting techniques described in Sambrook et al. (CIT. above). mRNA or cDNA generated on the matrix mRNA using the polymerase enzyme can be purified and separated by the method of gel electrophoresis. Amu is andnew acid in the gel is then subjected to blotting on a solid substrate, such as nitrocellulose. The solid substrate is treated with labeled probe and then washed to remove any dehybridization probes. Next, identify the resulting duplexes, including labeled probe. Usually, such a probe labeled with a radioactive component.
Brief description of figures
Figure 1-20 shows the data of the biochemical analysis obtained in the following examples and data sequencing for the coding part of the ORF 37, 5, 2, 15, 22, 28, 32, 4, 61, 76, 89, 97, 106, 138, 23, 25, 27, 79, 85 and 132. M1 and M2 correspond to molecular weight markers. Arrows indicate the position of principal of recombinant product or method, Western blotting) its position as a major immunoreactive band N.meningitidis. TR denotes the total protein extract N.meningitidis; OMV denotes the product of the outer membrane of the bubble N.meningitidis. In the description of the test the bactericidal action: mark diamondshows preimmune data; triangle sign (▴) denotes control data GST; circle (●) shows the data obtained with recombinant protein N.meningitidis. Computer analysis of the curve shows hydrophilicity (top)curve antigenic index (middle) and data analysis AMPHI (bottom). Computer program AMPHI used to predict the location of T-cell epitopes (Gao et al., 1989, J. Immunol., 143, 3007; Rberts et al., 1996, AIDS Res. Hum. Retroviruses, 12, 593; Quakyi et al., 1992, Scand. J. Immunol., suppl. 11, 9), and it is available from the software package Protean company DNASTAR Inc. (1228 S. Park str., Madison, WI 53715, USA).
The following examples describe the nucleotide sequences that were identified from N.meningitidis in parallel with presumably coded products broadcast, and the same sequence N.gonorrhoeae. He all of the nucleotide sequences are complete, i.e. they encode less than a full-sized protein of the wild type.
Examples of generally formed as follows:
- nucleotide sequence, which was identified from N.meningitidis (strain);
- product predskazateli broadcast from this nucleotide sequence;
data computer analysis of product broadcast on the basis of comparison existing databases;
- relevant gene and protein sequences identified from N.meningitidis (strain A) and N.gonorrhoeae;
- characterization of proteins, which indicate that they may have suitable antigenicity;
the results of biochemical analysis (expression, purification, ELISA, FACS, and the like).
Examples usually include details describing the level of identity between species and strains. Proteins, similar in its amino acid placenta is successive, similar in its structure and functions, i.e. the identity of the sequences often refers to the unity of origin in the phylogeny. Comparison of amino acid sequences of proteins with known function is widely used as a method for the assessment of intended functions of proteins with a new sequence, and, as was confirmed, was applied for the analysis of complete genomes.
Comparison of the sequences were performed in the system NCBI (http://www.ncbi.nlm.nih.gov using algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx & tBLASTx (see also, for example, Altschul et al., 1997, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucl. Acids Res., 25, 2289-3402). The search was conducted under the following programs polypeptide database: non-redundant sequence of GenBank+EMBL+DDBJ+PDB and GenBank+broadcast CDS+PDB+SwissProt+SPupdate+PIR.
To compare sequences of meningococcus and gonococcus used the tBLASTx algorithm, http://www.genome.ou.edu/gono_blast.html. The FASTA algorithm was used to compare the open coding framework ORF (GCG Wisconsin Package, version 9.0).
Mark points in the composition of the nucleotide sequences (e.g., at position 495 in the sequence SEQ ID NO: 11) determine the nucleotides that were randomly introduced with the aim of maintaining the coding frame. In the same way double underline corresponds to the deleted nucleotides. The article is full of letters (for example, in 496 m position in SEQ ID NO: 11) correspond to the uncertainties that arise when mapping independently carried out the sequencing reactions (some of the nucleotide sequences in the examples that follow are the result of a synthesis of two or more experiments).
Nucleotide sequences were scanned in all six coding framework with the goal of predicting the presence of hydrophobic domains, for which we used the algorithm based on statistical approaches Esposti et al., 1990, "Critical evaluation of the hydropathy of membrane proteins", Eur. J. Biochem., 190, 207-219). These domains represent a potential transmembrane regions or hydrophobic signal (leader) segments.
Open coding frames were predicted on the basis of fragmented nucleotide sequences, for which we used the program ORFFINDER (NCBI).
The underlined amino acid sequence indicates the presence of probable transmembrane domains or signal segments comprising the coding frame ORF in accordance with the predictions of the PSORT algorithm (http://www.psort.nibb.ac.jp). Functional domains were predicted using MOTIFS (GCG Wisconsin & PROSITE).
Various tests were used to assess the in vivo immunogenicity of the proteins identified in the examples. For example, proteins can be Express is arranged by recombinant and then used for screening of patient serum by the method of Western blot turns. A positive reaction between the protein and the serum of the patient indicates that the patient has previously formed the immune response to the analyzed protein, i.e. the protein is the immunogen. This method can be used to identify immunodominant proteins.
Recombinant protein can also be in the standard manner used for the preparation of antibodies, for example, in mice. They can be used for direct evidence that the protein is localized on the cell surface. Labeled antibody (e.g tagged with a fluorescent label for use in the method FACS) can be preincubation with intact bacteria - in this case the presence of the label on the surface of bacterial cells confirmed this localization of the corresponding protein.
In particular, the following methods (a to S) were used for expression, purification and biochemical analysis of the proteins of the present invention.
A) Obtaining chromosomal DNA
Meningococci N.meningitidis strain 2996 were grown to exponential growth phase in 100 ml of culture medium GC, collected by centrifugation and resuspendable in 5 ml buffer (20% sucrose, 50 mm Tris-HCl, 50 mm EDTA pH 8). After 10 minutes incubation on ice, bacteria were literally by adding 10 ml of lyse solution (50 mm NaCl, 2% sarcosine sodium, 50 mg/ml is proteinase) and the resulting suspension was incubated at 37° C for 2 hours. Conducted twice extraction with phenol (equilibrated to pH 8) and a single extraction with a mixture of chloroform and isoamyl alcohol (24:1). DNA precipitated with addition of 0.3 m sodium acetate and 2 volumes of ethanol and then collected by centrifugation. The resulting clot was washed once with 70%ethanol and re-dissolved in 4 ml of buffer (10 mm Tris-HCl, 1 mm EDTA, pH 8). The concentration of the resulting DNA preparation was determined by optical density at 260 nm.
B) the Design of the oligonucleotide
Synthetic oligonucleotide priming were designed based on the coding sequence of each coding frame ORF using (a) sequences of meningococcus (if available) or (b) A sequence of gonococci/meningococcus, adapted to the preferred codons of meningococcus (if this was necessary). Any anticipated signal segments are discarded, which generated the 5'-end amplication of the seed match the spot immediately after the alleged leader of the segment.
For most part of the ORF direct priming (5') included two site recognition by restrictase (BamHI/NdeI, BamHI/NheI or EcoRI/NheI, depending on your own restriction parameters of a specific gene); reverse (3') of the seed included a XhoI restriction-SAI is. This procedure was undertaken to clone each amplication product (corresponding to each coding frame ORF) in two different expression systems: pGEX-KG (using restriction sites or BamHI/XhoI or EcoRI/XhoI) and Reti+ (using restriction sites or NdeI/XhoI, or NheI/XhoI).
The end of the 5'-terminal priming:
The end of the 3'-terminal priming:
For frames ORF 5, 15, 17, 19, 20, 22, 27, 28, 65 and 89 were conducted by two independent amplification reaction with the aim of cloning each of the ORE in each of the two expression systems. Two different 5'-terminal priming were used for each ORF, despite the fact that inverse priming was the same 3'- XhoI seed, which is named above:
The end of the 5'-terminal priming:
The end of the 5'-terminal priming:
Codereuse the frame 76 was cloned using the expressing vector pTRC and expressed as chimeras with attached N-end polyhistidine "tail". In this particular case, the estimated signal segment were included in the final product. Restriction sites NheI/BamHI were introduced with the use of such seed:
The end of the 5'-terminal priming:
The end of the 3'-terminal priming:
As well as included the sequence recognizable by restrictase, the seed includes the nucleotides that hybridized sequence, which must be amplified. The number hybridizes nucleotides depended on the temperature of complete melting of the seed was determined for each seed using these formulas:
Tm= 4·(G+C) + 2·(A+T) (tail segment is excluded),
Tm= 64,9 + 0,41·(% GC) - 600/N (full seed).
The average melting temperature of the selected oligonucleotides were 65-70°for full oligonucleotides and 50-55°With separate parts hybridization.
Table 1 shows the direct and inverse seed was used for each amplification reaction. In some cases, it should be noted that the sequence specific priming does not meet fully the sequence coding for the setup portion of the frame. When conducting the initial amplification reaction, the complete 5'and / or 3'sequences were not known for some of meningococcal ORF, while the corresponding sequences were identified in the gonococcus. For amplification of the sequence of gonococci could therefore be used as a basis for designing a nucleating through sequence changes based on preference settings codons. In particular, we performed the following changes codons: ATA → ATT; TCG → TST; CAG → CAA; AAG → AAA; GAG → GAA; CGA → CGC; CGG → CGC; GGG → GGC. Such changes are reflected in table 1 by allocation of nucleotides in italics. It should be clear that once the full sequence, are identified, described this approach becomes no longer necessary.
Oligonucleotides were synthesized using DNA synthesizer/RNA model 394 firm Perkin-Elmer, suirvey column in 2 ml of NH4OH and exempt from protection by 5-hour incubation at 56°C. These oligonucleotides were precipitated with addition of 0.3 M sodium acetate and 2 volumes of ethanol. The samples are then centrifuged and the clots resuspendable either 100 μl or 1 ml of water. Was determined by optical density OD260using the spectrophotometer Lambda Bio (Perkin-Elmer), thereby evaluating the concentration, which is then delivered and to 2-10 pcmall/µl.
The standard PCR Protocol was as follows: 50-200 ng of genomic DNA was used as matrix in the presence of 20-40 μm of each oligonucleotide, 400-800 μm solution of a mixture of deoxyribonucleotides (dNTP), l × PCR buffer (including 1.5 mm MgCl2), and 2.5 units of DNA polymerase TaqI (using amplifier AmpliTaQ Perkin-Elmer, Gibco Platinum; DNA polymerase Pwo or Taq polymerase Tahara Shuzo).
In some cases, the PCR parameters were optimized by adding 10 ál of DMSO or 50 μl of 2M betaine.
After the "hot start" (adding polymerase to the mixture, preincubated for 3 minutes at a temperature of 95° (C) each sample was subjected to 2-step amplification: the first 5 cycles were performed using a hybridization temperature that is defined for oligonucleotides with excluded from the calculation of the "tail with restriction sites, followed by 30 cycles carried out at a temperature of hybridization, specific for full-length oligonucleotides. All cycles were completed the final 10-minute stages of completion chain with 72°C.
Standard cycles were as follows:
|The first 5 cycles||30 seconds|
|The final 30 cycles||30 seconds|
The duration of the stage of completion varied depending on the length of the coding frame ORF, the amplification of which is performed.
Amplification reaction was performed using gene-amplifier model 9600 or 2400 (Perkin-Elmer). To validate the results of the tenth part amplication volume was loaded in 1-1 .5%agarose gel and the size of each of the amplification products was compared with molecular weight DNA marker.
Amplified DNA was either loaded directly in a 1%gel or besieged ethyl alcohol and resuspendable in a suitable amount, which must be loaded in a 1%gel. The DNA fragment corresponding to the correct size band, then suirable and purified from the gel using a set of reagents Qiagen Gel Extraction Kit according to the manufacturer's instructions. The final volume of the DNA fragment was 30 ál or 50 ál in water or 10 mm Tris, pH 8.5.
D) Cleavage of PCR fragments
Purified DNA corresponding to amplificatoare slice was divided into two aliquots and subjected to double decomposition restrictable:
- NdeI/XhoI or Nhel/XhoI for cloning part 21b+ and far is her expression of protein in the form of a C-terminal part of the Chimera, including polyhistidine "tail";
- BamHI/XhoI or EcoRI/XhoI for cloning in the composition of the pGEX-KG and further expression of the protein in the form of the N-terminal part of the GST Chimera;
in the variant with the coding frame ORF76 NheI/BamHI for cloning in the composition of the vector pTRC-HisA and further expression of the protein in the form of the N-terminal part of the chimeras, including polyhistidine "tail";
- EcoRI/ > PST, EcoRI/SalI, SalI/ > PST to clone part of pGex-His and subsequent expression of the protein in the form of the N-terminal part of the chimeras, including polyhistidine "tail".
Each purified DNA fragment was incubated (at 37°C for 3 hours or over night) with 20 units each of restrictase (manufactured by New England Biolabs) at finite volume, or 30 μl or 40 μl in the presence of a suitable buffer. Split the product was then purified using a set of reagents QIAquick PCR purification kit according to the manufacturer's instructions and was suirable in a final volume of 30 μl or 50 μl of either water or 10 mm Tris-HCl (pH 8.5). The final concentration of DNA was determined by electrophoresis in 1%agarose gel in the presence of titiraupenga marker of molecular weight.
E) Processing the cloning vectors (retv, pGEX-KG, pTRC-HisA, pGex-His)
Double hits were subjected to 10 μg of plasmid using 50 units of each restrictase 200 μl reaction volume in the presence of a suitable buffer for n the Chi by incubation at 37° C. After downloading full of processed volume in 1%agarose gel, the band corresponding to the split restrictase vector was purified from the gel using a set of reagents QIAquick Gel Extraction kit (Qiagen) and DNA was suirable in 50 μl 10 mm Tris-HCl (pH 8.5). The DNA concentration was estimated by measuring OD260each sample and brought it to 50 mcg/ml 1 ál of each plasmid used in each procedure cloning.
The vector pGEX-His is a modified vector pGEX-2T, bearing area, which encodes six histidine residues located above the thrombin cleavage site and includes multiple cloning sites from the vector pTRC99 (Pharmacia).
The fragments corresponding to each coding frame, pre-treated restrictase and purified, were legirovanyh and part 22b, and the composition of the pGEX-KG. When the final volume of 20 µl molar ratio of 3:1 fragment and ligated vector using a 0.5 μl NEB DNA ligase of phage T4 (400 units/µl) in the presence of the buffer provided by the manufacturer. The reaction mixture was incubated at room temperature for 3 hours. In some experiments, ligation was carried out using a set of reagents Rapid Ligation Kit (Boehringer) according to the manufacturer's instructions.
The purpose of making a recombinant plasmids in approaching the s strain of 100 ál of competent cells of E. coli (strain DH5) were incubated with ligase reaction for 40 minutes on ice, then at 37°C for 3 minutes, then, after adding 800 ál of LB broth, again incubated at 37°C for 20 minutes. Cells are then centrifuged at maximum speed appendectomy microcentrifuge and resuspendable approximately 200 ál of the supernatant fraction. The resulting slurry is then sown on LB medium containing ampicillin (100 mg/ml).
Screening of recombinant colonies was carried out by growing the five randomly selected colonies overnight at 37°With either 2 ml (pGEX clones or RTS)or 5 ml (clones pet) of LB broth + 100 μg/ml ampicillin. Cells were then palletizable and DNA was extracted using the assay kit QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer's instructions to a final volume of 30 µl. 5 µl of each individual minipreparation (approximately 1 g) were treated with restrictase or NdeI/XhoI or BamHI/XhoI and fully processed volume was loaded in 1-1 .5%agarose gel (depending on the expected size of the insert) along with molecular weight marker (1Kb DNA Ladder, Gibco). Screening of positive clones was carried out according to the parameters of the correct size for the desired insert.
For cloning the coding framework ORF 110, 111, 113, 115, 119, 122, 125 and 130 double-processed restrictase PCR product is ligated into the composition of the double-treated restrictase vectors using cloning EcoRI/ > PST sites in the case of framework ORF 115 and 127 - EcoRI/SalI sites, and in the case of frame 122 - SalI/ > PST -sites. After cloning, the recombinant plasmids were introduced into cells of E. coli strain W3110. Individual clones were cultured overnight at 37°in L-broth with the addition of 50 μl/ml ampicillin.
Each coding frame ORF cloned into a composition expressing vector was used for transformation of strain suitable for expression of the recombinant protein product. 1 ál of each construct used for transformation 30 μl of E. coli strain BL21 (vector pGEX), E. coli strain TOP10 (vector pTRC) or E. coli strain BL21-DE3 (vector pet) as described above. In the case of vector pGEX-His the same E. coli strain E. coli (W3110) was used for initial cloning and expression. Single recombinant colonies were sown in 2 ml culture of LB broth with ampicillin (100 μg/ml), incubated over night at 37°C, then diluted in the ratio of 1:30 in 20 ml of LB with ampicillin (100 μg/ml) in 100-ml flasks, checking out with the optical density OD600that should be 0.1 to 0.15. These flasks were incubated at 30°C in a rotary shaker water bath until, when the optical density will indicate the output of the culture in the exponential phase of growth, suitable for the induction of expression (0,4-0,8 OD for vectors pet and TRC; 0,8-l OD for pGEX vectors and pGEX-His). For vectors pet, pTRC and pGEX-His protein expression was induced by adding 1 mm IPTG, while in the case of vector pGEX the final concentration of IPTG was 0.2 mm. After 3 hours incubation at 30°With the final concentration of the sample was checked largest OD. To control the expression of 1 ml of each sample was removed, centrifuged in microcentrifuge received clot resuspendable in phosphate-buffered saline (FSB) and analyzed by the method of SDS-electrophoresis in 12%SDS page with staining of Kumasi blue. The full amount of the sample was centrifuged at 6000g and the resulting clot resuspendable the FSB for future use.
H) large-Scale purification of recombinant GST-proteins
A single colony was cultured overnight at 37°C in LB medium with ampicillin agar plate. Bacteria were sown in 20 ml of LB with ampicillin in liquid culture in a shaker containing a water bath, and were cultured overnight. Bacteria were diluted 1:30 in 600 ml of fresh culture medium and left to grow at optimum temperatures (20-37° (C) to achieve an optical density OD5500,8-1. Expression of protein was induced by adding 0.2 mm IPTG and subsequent 3-hour incubation. The culture was centrifuged at 8000 rpm at 4°C. the Supernatant fraction was removed and the clot bacterial glue is OK resuspendable 7.5 ml of cold phosphate-saline buffer. Cells were destroyed by ultrasound on ice for 30 seconds at a power of 40 W with use of the device In-15 (Branson), was twice frozen and thawed and centrifuged. The supernatant fraction was collected and stirred in 150 µl glutationozalezny-4B resin (Pharmacia) (pre-washed in the FSB), and incubated at room temperature for 30 minutes. The sample was centrifuged at 700g for 5 minutes at 4°C. the Resin was washed 2 times with 10 ml of cold FSB within 10 minutes, resuspendable in 1 ml of cold FSB and downloaded the free column. The resin was washed twice in 2 ml of cold FSB as long as the flowing material has reached an optical density OD2800,02-0,06. Chimeric GST-protein was suirable by adding 700 ál of cold glutathione-lucynova buffer (10 mm reduced glutathione, 50 mm Tris-HCl) and fractions were collected before reaching OD280value of 0.1. 21 μl of each fraction were loaded in 12%SDS-gel using either molecular standards for SDS-page from Biorad (M1) (200, 116,25, 97,4, 66,2, 45, 31, 21,5, 14,4, 6,5 KD), or set of standards from Amersham (M2) (220, 66, 46, 30, 21,5, 14,3 KD). Despite the fact that the molecular mass of GST is 26 KD, this value should be added to the value of the molecular weight determined for each chimeric GST-containing protein.
I) Analysis of the solubility of His-chimeras (coding frame 111-129)
For the analysis of R is stoimosti His-chimeric products of expression of the clots 3-ml cultures resuspendable in buffer M1 (500 μl FSB pH of 7.2). Added 25 μl of lysozyme and bacteria were incubated for 15 minutes at 4°C. the resulting clot was irradiated with ultrasound for 30 seconds at a power of 40 W using a Branson ultrasonic setup B-15, twice frozen and thawed and then re-divided into the clot and the supernatant fraction by centrifugation. The supernatant fraction was collected and the remaining clot resuspendable buffer M2 (8 M urea, 0.5 M NaCl, 20 mm imidazole and 0.1 M NaH2PO4) and incubated for 3-4 hours at 4°C. After centrifugation the supernatant fraction was collected and the clot resuspendable in M3 buffer (6 M guanidine-HCl, 0.5 M NaCl, 20 mm imidazole and 0.1 M NaH2PO4) overnight at 4°C. the Supernatant fraction at each of these stages were analyzed by electrophoresis in SDS-page.
Proteins expressed by a part of the ORF 113, 119 and 120, are found to be soluble in phosphate-buffered saline, while the products are within the ORF 111, 122, 126, 129, demand for dissolution of the urea, and products within the ORF 125 and 127 - handinhand.
J) large-Scale purification of His-Chimera
A single colony was cultured overnight at 37°C in LB medium with ampicillin agar plate. Bacteria were sown in liquid culture in 20 ml LB medium with ampicillin, and incubated overnight in a shaker water bath. BA the bacteria were diluted 1:30 in 600 ml of fresh medium and left to grow at optimum temperatures (20-37° (C) to the density OD550of 0.6-0.8. Expression of protein was induced by adding 1 mm IPTG and the culture is then incubated for three hours. Then the culture was centrifuged at 8000 rpm at 4°With, removing the supernatant layer and bacterial clot resuspendable in 7.5 ml of either (1) cold buffer A (300 mm NaCl, 50 mm phosphate buffer, 10 mm imidazole pH 8) for soluble proteins, or (2) buffer (8 M urea, 10 mm Tris-HCl, 100 mm phosphate buffer pH 8.8) for insoluble proteins.
Cells were destroyed by ultrasound on ice for 30 seconds at a power of 40 W using a Branson ultrasonic setup B-15, twice frozen, thawed and re-centrifuged.
In the case of insoluble proteins in the supernatant layer was stored at -20°With, while clots resuspendable in 2 ml of buffer (6 M guanidine hydrochloride, 100 mm phosphate buffer, 10 mm Tris-model HC1 - pH 7.5) and treated in a homogenizer for 10 cycles. The resulting product was centrifuged at 13000 rpm for 40 minutes.
The supernatant fraction was collected and mixed with 150 μl of Nickel-containing (Ni2+) resin (Pharmacia) (pre-washed with buffer a or buffer) and incubated at room temperature with thorough stirring for 30 minutes. The sample was centrifuged at 700g for 5 minutes at 4°C. the Resin washed twice in 10 ml puff the RA And or within 10 minutes, resuspendable in 1 ml buffer a or b, and downloaded the free column. The resin was washed either (1) in 2 ml of cold buffer And at 4°or (2) at room temperature in 2 ml of buffer up until the flowing material will not reach the optical density OD2800,02-0,06.
The resin was washed either in (1), 2 ml of cold buffer with 20 mm imidazole (300 mm NaCl, 50 mm phosphate buffer, 20 mm imidazole pH 8), or (2) buffer D (8 M urea, 10 mm Tris-model HC1, 100 mm phosphate buffer pH 6.3) up until the flowing material will not reach the optical density OD2800,02-0,06. Recombinant His-protein was suirable by adding 700 μl of either (1) cold lucynova buffer A (300 mm NaCl, 50 mm phosphate buffer, 250 mm imidazole pH 8), or (2) lucynova buffer (8 M urea, 10 mm Tris-HCl, 100 mm phosphate buffer pH 4.5) and the fractions obtained were selected to density OD280=0,1. 21 μl of each fraction were loaded in 12%SDS-gel.
To) Resaturate His-chimeric proteins
To denaturirovannyj proteins was added 10% glycerol. Proteins were then diluted to a concentration of 20 µg/ml using dialysis buffer I (10% glycerol, 0.5 M arginine, 50 mm phosphate buffer, 5 mm reduced glutathione, 0.5 mm oxidized glutathione, 2 M urea at pH 8.8) and were dialyzed against the same buffer at 4°C for 12-14 hours. Further, this protein were dialyzed against cialisno what about buffer II (10% glycerol, 0.5 M arginine, 50 mm phosphate buffer, 5 mm reduced glutathione, 0.5 mm oxidized glutathione, pH 8.8) for 12-14 hours at 4°C. the protein Concentration was determined using the following formula:
Protein (mg/ml) = (1,55 × OD280) - (0,76 × OD260)
L) large-Scale purification of His-chimeras (coding frame ORF 111-129)
500 ml bacterial cultures were incubated and chimeric proteins were obtained soluble in buffers M1, M2 or M3, for which we used the protocols described above. The crude extract of bacteria were loaded on superfast column of Ni-NTA (Quiagen), equilibrated with buffer M1, M2 or M3, depending on in which of these buffers soluble chimeric protein. Unbound material was suirable washing the column with the same buffer. The specific protein was suirable using an appropriate buffer containing 500 mm imidazole and were dialyzed against the appropriate buffer, which did not contain the imidazole. After each flow column was recovered by washing with at least two column volumes of 0.5 M sodium hydroxide and re-equilibration before the next use.
M) Immunization of mice
For immunization of mice by intraperitoneal injection was used at 20 μg each of the purified proteins. In cases where the coding framework ORF 2, 4, 15, 22, 27, 28, 37, 76, 89 and 97, when immunization m the necks of Balb/C on the 1st, 21st and 42nd days of adjuvant used aluminum hydroxide, and the immune response was monitored in samples on day 56. In the case of coding frames ORF 44, 106 and 132 mice of CD1 was immunizable in the same Protocol. In the case of coding frames ORF 25 and 40 CD1 mice were immunizable in the same Protocol, replacing the adjuvant aluminum hydroxide in Freund adjuvant; the difference was that the immune response was monitored on 42nd and not on the 56th day. Similarly, in cases where the coding framework ORF 23, 32, 38 and 79 CD1 mice were immunizable with Freund adjuvant, but the immune response was determined on the 49th day.
N) Test TYPHUS (serum)
The decapsulated MenB strain M7 were sown on chocolate agar plates and incubated over night at 37°C. Bacterial colonies were picked from agar plates using a sterile brush and sown in 7 ml of culture broth Mueller-Hinton (Difco)containing 0.25% glucose. Bacterial growth was monitored every 30 minutes largest OD620. The bacteria were left to grow until reaching an optical density OD of 0.3-0.4. The culture was centrifuged for 10 minutes at 10000 rpm the Supernatant fraction was removed and the bacteria were washed once with phosphate-saline buffer, resuspendable the FSB, containing 0.025% formaldehyde, and incubated for 2 hours at room temperature and then eseva, over night at 4°C. 100 μl of bacterial cells was added to each well of 96-hole Gamerscore tablet and incubated over night at 4°C. Then the wells are washed three times washing with phosphate twin-buffer (0.1% tween-20 in phosphate-buffered saline). To each well was added 200 μl of saturating buffer (2.7% of polyvinylpyrrolidone-10 in water) and the plates were incubated for 2 hours at 37°C. the Wells were washed three times in FSB. To each well was added 200 μl of diluted serum (dilutional buffer: 1% bovine serum albumin, 0.1% tween-20, 0.1% of NaN3the FSB) and the plates were incubated for 90 minutes at 37°C. Then the wells are washed three times in FSB. To each well was added 100 μl conjugated with horseradish peroxidase rabbit antimachine serum (Dako)diluted 1:2000 in dilutional buffer, and the plates were incubated for 90 minutes at 37°C. To each well was added 100 μl of substrate for horseradish peroxidase buffer (25 ml citrate buffer pH 5; 10 mg O-phenyldiamine and 10 ál of water) and the tablets were left at room temperature for 20 minutes. To each well was added 100 μl of sulfuric acid and was determined by OD490. The TYPHOID test was positive when the OD value4902.5 times the corresponding parameter preimmune serum.
(O) Procedure test FACScan binding is of aktery
The decapsulated MenB strain M7 were sown on chocolate agar plates and incubated over night at 37°C. Bacterial colonies were collected from agar plates using a sterile brush and were sown in 4 test tubes, each containing 8 ml of the culture broth Mueller-Hinton (Difco)containing 0.25% glucose. Growth of bacteria was monitored every 30 minutes by OD620. Bacteria were left to grow until reaching an optical density value of 0.35 to 0.5. The culture was centrifuged for 10 minutes at 4000 rpm the Supernatant fraction was removed and the clot resuspendable in blocking buffer (1% bovine serum albumin, 0.4% of NaN3) and centrifuged for 5 minutes at 4000 rpm Cells resuspendable in blocking buffer before reaching OD620value of 0.07. In each well of 96-hole tablet Costar was added 100 μl of bacterial cells. To each well was added 100 μl of diluted (1:200) serum (blocking buffer) and the plates were incubated for 2 hours at 4°C. the Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant was aspirated layer and the cells were washed by adding to each well 200 μl of blocking buffer. To each well was added 100 μl of goat antimelanoma antibody F(ab)2conjugated with R-phycoerythrin and diluted 1:100, and the plates were incubated for 1 the Asa at 4° C. the Cells were besieged by centrifugation at 4000 rpm for 5 minutes and washed by adding to each well 200 μl of blocking buffer. The supernatant was aspirated layer and cells resuspendable 200 ál FSB at each well + 0.25% formaldehyde. The sample was transferred into a test tube for FACScan (sorting cells with induced fluorescence) and read them. Conditions FACS-scan were as follows: FL1 - enabled; FL2 and FL3 - off; FSC-H threshold value 92; FSC RMT - voltage E-02; SSC RMT - 474; amplitude growth at 7.1; FL-2 RMT - 539; a compensation value is 0.
R) Receiving OMV
Bacteria were cultured overnight on five plates GC, collected by loop and resuspendable in 10 ml of 20 mm Tris-HCl. Inactivation by heating was carried out for half an hour at 56°and bacterial cells are destroyed by ultrasound for 10 minutes on ice (50% load in the cycle of irradiation, 50% power output). Intact cells were removed by centrifugation at 5000g for 10 minutes and the fraction of cell membranes were isolated by centrifugation at 50000g for 75 minutes at 4°C. For the extraction of cytoplasmic membrane proteins from a crude fraction of the external membranes full faction resuspendable 2%sarkosyl (Sigma) and incubated at room temperature for 20 minutes. The suspension was centrifuged at 10000g during the course the e 10 minutes in order to remove aggregates and the supernatant fraction was then subjected to ultracentrifugation at 50000g for 75 minutes getting a bunch of external membranes. The outer membrane resuspendable in 10 mm Tris-HCl (pH 8) and the protein concentration was determined in the test Bio-Rad Protein using as a standard of bovine serum albumin.
Q) Obtaining full extracts
Bacteria were cultured overnight on the disc, GC, collected by loop and resuspendable in 1 ml of 20 mm Tris-HCl. Inactivation by heating was carried out for 30 minutes at 56°C.
R) Western blotting
Purified proteins (500 ng per track), outer membrane vesicles (5 g) and total cell extracts (25 μg)derived from cells of strains of MenB-2996, was loaded onto 15%polyacrylamide gel for SDS-electrophoresis and transferred to nitrocellulose membrane. The transfer was carried out for 2 hours at 150 mA at 4°in a special buffer (0.3% of the main Tris, of 1.44% glycine, 20% methanol). Membranes were saturated by incubation overnight at 4°in saturating buffer (10% skimmed milk, 0.1% Triton X-100 FSB). Membranes were washed twice in wash buffer (3% skimmed milk, 0.1% Triton X-100 FSB), and incubated for 2 hours at 37°With mouse serum diluted to 1:200 in wash buffer. Membranes were washed twice and incubated for 90 minutes with a labeled horseradish peroxidase artemisinin immunoglobulin diluted to 1:2000. Membranes were washed twice with 0.1% Triton X-100 FSB and processed using is a substrate W from a set of reagents Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.
S) Test for bactericidal
Cells of strain MC were cultured over night at 37°on chocolate agar plates. 5-7 colonies were selected and used for sowing in the culture broth Mueller-Hinton (7 ml). Suspensions were incubated at 37°in nutator and left to grow until reaching OD620values of 0.5-0.8. Each culture was divided into aliquots in sterile 1.5-ml appendrows tubes and centrifuged for 20 minutes at maximum speed microcentrifuge. The resulting clot was washed once in buffer Gay (Gibco) and resuspendable in the same buffer to the value OD6200,5, diluted in buffer Gay to 1:20000 and kept at 25°C.
In each well of 96-well plate to the tissue culture medium was added 50 μl of buffer Gay in + 1% bovine serum albumin. To each well was added 25 μl of diluted mouse serum (1:100 buffer Gay + 0,2% bovine serum albumin) and the plate is incubated at 4°C. To each well was added 25 μl of the above suspension of bacterial cells. To each well was added 25 μl of either inactivated by heating (up to 56°C in a water bath for 30 minutes), or a normal complement rabbits. Immediately following the addition of complement rabbits and 22 µl of each treatment is CA (holes) were sown on agar plates Meuller-Hinton (time 0). 96-well plates were incubated for 1 hour at 37°during the rotation and then to 22 μl of each sample (wells) were sown on agar plates of Mueller-Hinton (time 1). After incubation overnight counted colonies, the corresponding time segments 0 and 1 (hours).
Table II summarizes data on the cloning, expression and purification.
The following partial DNA sequence was identified from N.meningitidis (SEQ ID NO: 1):