The selected dna sequence, a vector, a method of obtaining a homogeneous gp350 protein, homogeneous gp350 protein, a pharmaceutical composition for the treatment of ebv-associated disease or condition

 

(57) Abstract:

The proposed selected DNA encoding a protein gp350. Protein gp350 used in pharmaceutical compositions for the treatment of EBV-associated disease or condition. The selected DNA is part of the vector, which is introduced into a cell of the host, where the expression of gp350 protein. DNA in the vector eliminates the production of protein gp220 and produces only gp350. 6 C. and 2 h. p. F.-ly, 5 Il. 3 table.

The virus of Epstein-Barr (Epstein-Barr= EBV), a member of the group of herpes viruses that may cause people infectious mononucleosis. This disease affects more than 90% of the population. Analysts of health estimate losses associated with this disease in the United States, 100 million dollars annually. This virus is spread mainly by droplets from the saliva of individuals that distribute the virus. Children infected with EBV, in most cases, asymptoticly or they have very mild symptoms, whereas in adolescents or adults with the infection develop typical infectious mononucleosis, characterized by fever, pharyngitis and adenopathy. People who were infected, anti-EBV antibodies persist throughout the rest of life, and p. vaccines.

In addition to its infectious characteristics, it has been shown that EBV transforms lymphocytes in rapidly dividing cells, and therefore indirectly associated with various lymphomas, including lymphoma Burkitts or oral the hairy leukoplakia. EBV were also detected in tissue samples from individuals with nasopharyngeal tumors. Worldwide, there are about 80,000 cases of cancer of the nasopharynx, and it is prevalent among ethnic Chinese.

Development of a live, attenuated vaccine for EBV was and remains a serious problem. Due to the potential carcinogenic nature associated with EBV, the researchers objected to the use of approaches associated with live vaccines. This invention overcomes the problems associated with live vaccines is, by creating methods and compositions for subunit vaccines that do not require the use of potentially carcinogenic live virus. Subunit vaccine represents one or more of the antigenic proteins of the virus, which cause an immune response and develop immunity.

Two of the most important EBV antigenic proteins are glycoproteins Dr/300 and Dr/200, which form part of the membrane of the viral membrane and allows the protein to cell membranes, CD21. Cm. Nemerow, J. Virology 61: 1416 (1987). They were marked as candidates for subunit vaccines, but the difficulties in obtaining active antigenic protein isolated from natural sources, and low outputs of glycoproteins in obtaining them from recombinant sources will complicate attempts of researchers and vaccine development. In the literature indicate that these proteins have a wide range of molecular weights (350 or 300 kilodaltons /CD/ for one of the proteins and 220 or 200 kilodaltons to another protein). Here the protein Dr or 300 is referred to as Dr and protein 220 or 200 here referred to as protein Dr. In General, both of these proteins here designated as Dr/220 protein (proteins).

Alternative splanirovany, a single gene encodes Dr/220 proteins and leads to the creation of Dr and Dr mRNA transcripts; and unknown natural variants of splicing sites of the gene Dr/200. The gene products of the two products of expression are Dr and Dr proteins. The open reading frame for Dr/220 DNA sequence is 2721 couple of reasons (b). All reading frame encodes 907 amino acids Dr. Cm. U.S. patent 4707358 issued Kieff (1987). Splanirovany variant reading frame covers 2130 reason is Dr protein is 95 KD and 70 KD, respectively. The measured molecular weight expressed Dr protein and Dr protein vary, but are approximately 350 kilodaltons and 220 kilodaltons, respectively. Intensive glycosylation of proteins is responsible for the differences between the predicted and actual molecular weights. In each of the individual cells of both protein Dr and Dr are produced in a molar ratio from about 6: 1 to 1: 1. For example, in cells of the B95-8, which persistently infected with EBV, this relationship seems to vary, but sometimes reaches values of 6: 1. Cm. Miller, Proc. Natl. Acad. Sci 69: 383 (1972).

Similarly, recombinant obtaining these glycoproteins therefore usually leads to a mixture Dr and Dr proteins. Still Dr/220 proteins expressed in cells of rat pituitary cells in the ovary of the Chinese hamster VERO, kidney, African green monkeys, as well as in yeast cells. Cm. Whang, J. Virol. 61: 1796 (1982), Motz, Gene 44: 353 (1986) and Emini Virology 166: 387 (1988). The expression system of the virus bovine papilloma was used to obtain Dr/220 protein in mouse fibroblastic cells. Cm. Madej, Vaccine 10: 777 (1992). Laboratory and vaccine strains of vaccinia virus were used for the expression of Dr/220 protein. Modified Ryoko is Benante truncated constructs gp350/220 gene, does not contain connecting with membrane sequences. Such designs are still produced by the mixture of two Dr and Dr, but the deletion of the binding site with the membrane allows the secretion of the protein. Cm. Finerty, J. Gen. Virology 73: 449 (1992) and J. Madej Vaccine 10: 777 (1992). In addition, they also received various recombinante obtained restriction fragments and fusion proteins containing various gp350/220 sequence, and they were expressed in E. coli. Cm. EP patent publication 0173254 published July 24, 1991

Accordingly, studies of EBV related Dr/220, to date have been focused on obtaining effective expression of native Dr/220 sequence or modifitsirovannoi sequence that does not contain the transmembrane domain, which resulted in the receipt of a mixture of two alternative playerowner variants of native or not containing the transmembrane domain of the protein, or upon receipt of the epitope sequences of the fragment in-galactosidase proteins merge.

Known partially purified preparations Dr/220. Cm. Finerty, J. Gen. Virology 73: 449 (1992) (recombinante received, partially purified). As for native gp350/220 protein, in most cases, the process to clean the subunit vaccine. However, highly purified preparations of antigenic active gp350 protein from native (i.e. not recombinant) sources, yet were obtained, as reported in the literature. Cm. David, J. Immunol Methods 108: 231 (1988). In addition, the recombinant virus vaccines expressing gp350/220 protein was used for immunization khlopchatnika Tamarin against induced EBV lymphoma. Cm. J. Med. Virology 25: 189 (1988), Mackett, EMBO J. 4: 3229 (1985) and Mackett, VACCINES 86, PP 293 (Lerner RA, Chanock RM, Brown F (Eds 1986, Cold Spring Harbor Laboratory). However, until now, was not constructed virus gp350/220 DNA sequence in order to ensure the expression of one or the other separately splanirovano variants of the gene, thereby producing a clean gp350 or Dr protein. Were not constructed recombinant or mutant viruses that expressed would be one or the other of gp350 and Dr proteins.

Usually splicing sites facilitate the processing of pre-mRNA molecules in the mRNA. The virus polyoma splicing sites are required for efficient accumulation of subsequent mRNA. Change 3' and 5' splicing sites in the transcripts of the virus polyoma reduces or completely blocks the accumulation of mRNA. Cm. Treisman Nature 292: 595 (1981). In the SV40 virus excised introns facilitate transscout binding of small nuclei, RNP particles, believe that presbyteriana mRNA may be unable to find a way to be in the design of processing. It has been shown that point mutations in sigam splicing of the exon/intron reduce the splitting of the exon/intron and can disrupt pre-mRNA processing, nuclear transport, and stability. Cm. Ryu, J. Virology 63: 4386 (1989) and Gross, Nature 286: 634 (1980). Therefore, until the present invention, the influence of modification of splicing sites on the functional expression and antigenic activity of proteins encoded by EBV gp350/220 sequence, it was completely unknown and unpredictable.

Additional known on this issue, the literature include the following. EBV biology and disease in the General plan are listed in the overview Straus, Annal of Int. Med. 118: 45 (1993). Description EBV BLLFI open reading frame is shown Baer, Nature 310: 207 (1984). Description DNA and amino acid sequences of gp350/220 virus Epstein-Barr provided in article Beisel, J. Virology 54: 665 (1985) and Biggin, EMBO J. 3: 1083 (1984) and in U.S. patents 4707358 issued Kieff et. al (1987). Comparison of DNA sequences encoding gp350/220 virus Epstein-Barr types a and b, are disclosed in Lee, Virology 195: 578 (1993). Monoclonal antibodies that exhibit neutralizing activity against gp350/220 glycollate for donor and acceptor splicing sites disclosed the Mount, Nucleic Acids Res 10: 459 (1982).

In one aspect of the present invention proposed a non-splicing variants of EBV gp350/220 DNA sequence. The DNA sequence of the present invention may include a selected DNA sequence that encodes the expression of a homogeneous gp350 protein. The DNA sequence encoding gp350 protein, characterized as a sequence containing identical or almost identical to the nucleotide sequence of Fig. 1, where the native nucleotide sequence of the donor and acceptor splicing sites replaced by negativnye nucleotides and fragments thereof. The DNA sequence may include 5' and 3' non-coding sequences flanking the coding sequence, and further include aminoterminal signal sequence. Fig. 1 illustrates a non-coding sequence and indicates the end of the proposed signal sequence with an asterisk. However, it is obvious that the DNA sequence of the present invention may not contain some or all of these flanking sequences or signal sequences. The DNA sequence of the non-splicing variants of this izobreteniya splicing sites of the gene, coding gp350/220/. This eliminates the production of gp220 protein, which is produced only gp350 protein.

Accordingly, another aspect of the invention includes a homogeneous gp350 proteins and methods for producing proteins by expression of non-splicing variants of EBV gp350/220 DNA sequence in the cells of the respective prokaryotic or eukaryotic hosts. As regards the terms used in relation to gp350 protein, homogeneity means the complete or almost complete absence of gp220 protein. We note that the homogeneous gp350 protein, recombinante obtained in mammalian cells or insect, as far as we know, has never been mentioned so far in the scientific literature.

In another aspect of the proposed homogeneous gp350 proteins containing additional deletions in secretroom product. Such deletions include either removal of the transmembrane region or deletion of the transmembrane segment and the rest of the C-end of gp350. Such additional modified DNA sequence and coded for proteins constitute another aspect of the invention.

Also proposed recombinant DNA molecule containing vector DNA and a DNA sequence, codrului regulatory sequence, able to manage the replication and expression of a homogeneous gp350 in the cells of the selected owner. The host cells transformed with such DNA molecules for use in the expression of recombinant homogeneous gp350, also provided in this invention.

DNA molecules and transformed cell hosts in the present invention is used in another aspect of the invention, a new method of obtaining recombinant homogeneous gp350 protein or its fragments. In this way the cell line transformed by DNA sequence that encodes a homogeneous gp350 protein or its fragment (or the above-described recombinant DNA molecule), operatively linked to a suitable regulatory or controlling the expression of a sequence capable of controlling expression of the protein in cultured under appropriate conditions that ensure the expression of recombinant DNA. Then expressed proteins harvested from the host cells or culture medium by conventional means. As host cells in this way you can use a number of known cells; currently preferred mammalian cells and insect cells.

DNA sequence and p is containing EBV antigenic determinants. Such compounds find use in subunit vaccines for the prevention, treatment and prevention of diseases associated with EBV, as mononucleosis, lymphoma Burkett and carcinoma of the nasopharynx. Accordingly, in another aspect of the present invention presents such therapeutic and/or immunogenic pharmaceutical composition for the prevention and treatment of these associated with EBV conditions and diseases in humans, as infectious mononucleosis, lymphoma Burkett and carcinoma of the nasopharynx. Such therapeutic and/or immunogenic pharmaceutical composition include immunogen inducing effective amount of one or more of the homogeneous gp350 proteins of the present invention in a mixture with a pharmaceutically acceptable carrier, such as aluminum hydroxide, saline or phosphate superyoung saline solution, as is known in the art. Under "immunogen inducing" means the amount which is sufficient to stimulate the mammal the production of antibodies to EBV. In another embodiment, the active ingredient can be entered in the form containing liposomes units. For prophylactic use of such pharmaceutical compositions metatony to promote education in the patient of antibodies; and re-vaccinated after six months or a year.

The following aspect of the invention, a method of treatment associated with EBV diseases and conditions due to the introduction of the patient, in particular person, immunogen inducing therapeutically effective amount of a homogeneous gp350 protein in a suitable pharmaceutical carrier. Another method of the invention is a method of stimulating the immune response against EBV due to the introduction of the patient immunogen inducing effective amount of a homogeneous gp350 protein in a suitable pharmaceutical carrier.

Fig. 1 illustrates the DNA and amino acid sequence of gp350/220 (Beisel, J. Virology 54: 665 (1985)). Specified donor and acceptor splicing sites. Transmembrane section are marked with vertical arrows, and the asterisk (*) marked the end of a presumptive signal sequence. The numbering of nucleotides to the left; the numbering of the amino acids on the right.

Fig. 2 illustrates the construction of gp350 deletions and site-directed mutants. Plasmid maps, marked as pMDTM and pMSTOP, are examples of non-splicing variants of gp350/220 of the present invention. In Fig. 2A linear model gp350 crack and the other signal sequence (SS) and transmembrane domain (TM), while the chart of the gene represented an important restriction sites. gp350 gene was cloned in two segments, HindIII/BfaI BLSH1 fragment and BanI/HindIII BLSH2 fragment. SCYT create using polymerase chain reaction, starting with the specified plot BLLF1. Fig. 2B illustrates the cloning scheme for pDTM, pSTOP, pMDTM and pMSTOP (plasmids are not to scale). Details of the scheme of the cloning described in examples 1 and 2. For plasmid maps contain appropriate restriction sites used in cloning vectors and gp350 gene fragments. Mutations of splicing sites in pMDTM and pMSTOP marked with asterisks.

Fig. 3 illustrates the results of immunosurgery homogeneous gp350 protein from pMDTM clones in the analysis of SDS-PAGE. Positive control cells (GH319), secreting a truncated form of gp350/220 protein, negative control cells (REE) and some pMDTM clones metabolically labeled35S methionine for 5.5 hours; homogeneous gp350 protein was immunogen of the supernatant liquids tissue cultures. For each cell type samples of the supernatant fluid tissue culture (S) and precipitated gp350/220 (IP) were treated by electrophoresis on 5% SDS-PAGE (polyacrylamide gel electrophoresis). On the left is a marker of molecular weights.

Fig. the but in the examples 3, 4.

Disclosed are compositions and methods that include cloned EBV DNA sequence encoding a non-splicing variants gp350 protein. As mentioned, such a non-splicing variants here are referred to as homogeneous gp350 proteins. Usually, if the gp350/220 gene is expressed in mammalian cells, produced two gene product, gp350 and gp220, due to RNA splicing of the gene. The present invention provides the ability to get only one gene product, gp350. The present invention involves removal of some or all RNA splicing site of signals in gp350 gene and gene expression in cells of a suitable host. Mutations gp350/220 gene injected to prevent the production of 220 CD version of the protein, when gp350/220 Express in mammalian cells. The result of mRNA transcripts encoding only gp350. The exception gp220 expression through the use of gp350/220 non-splicing variant gp350/220 gene leads to increased production of gp350 compared to gp220. The production of gp220 not essential for producing effective anti-EBV vaccine, as gp350 contains all potentially antigenic sites that are on the gp220.

Therefore, in one aspect of the present invention obespechivayuschim, what codon donor splicing site, encoding a 501 amino acid, and codon acceptor splicing site that encodes the amino acid 698, were modified by replacing the native nucleotides negativnye nucleotides. Preferably, the native nucleotides to replace negativnye nucleotides so that the amino acid sequence remained the same. For example, native nucleotides AAGT in donor splicing site (nucleotides 1500-1504) and native nucleotides a and T, flanking GG acceptor splicing site (nucleotides 2091 and 2094), substituted nucleotides GTCA and T and A, respectively. Accordingly, as a result of such substitution in the donor site of the glutamine at amino acid position 500 and the serine at position 501 remain the same. Similarly, in the modification in the acceptor site of threonine at position 697 amino acids and glycine at position 698 remain the same.

Similarly, substitution differ from those given in the example can be easily carried out by specialists, as further set forth herein.

Therefore, in one aspect of the present invention includes a homogeneous gp350 proteins. Homogeneous gp350 proteins are distinguished further by the fact that mo is about 907, with amino acids 1 to 862 or amino acids 1 to 907, excluding amino acids 863-881, each of which may contain or not contain N-terminal signal sequence of 18 amino acids. In addition, the proposed analogues of homogeneous gp350 proteins and these include mutants in which there are variations in amino acid sequences that retain Athenry activity, and, preferably, homologous, at least 80%, more preferably 90% and even more preferably 95%, with a corresponding plot homogeneous gp350 proteins. Examples include proteins and polypeptides with minor amino acid variations in the amino acid sequence shown in Fig. 1; in particular, conservative substitutions of amino acids. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains. Genetically coveraee amino acids are generally divided into 4 families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Vanilla is to revive, individual replacement of leucine or a similar conservative replacement of an amino acid structurally related amino acid will not have a significant effect on the antigenic activity or functionality.

The present invention offers an advantage over simple selection gp350. As gp350 and gp220 have the same biochemical properties, gp220 often allocated together with gp350 when cleaning preparations. Cellular expression of only non-splicing variant gp350/220 gene facilitates selection of the protein. This reduces the cost of obtaining gp350. The present invention also makes it easier to obtain biochemical characteristics of the initial material for selection gp350. Due to the presence of only one type of analysis of the protein and analysis of amino acid sequences can be accomplished without taking into account the presence of the second kind.

The present invention additionally offers the advantage of enhanced production gp350 protein. Preventing splicing gp350 gene shifts the production by the cell of the two products (gp350 and gp220) on the production of only gp350. In some cells, as estimated concentrations gp220 amount of 30-100% of gp350 concentration. When excluding splashiest gp350/220 gene described Beisel, J. Virology 54: 665 (1985) and Biggin, EMBO J. 3: 1083 (1984) and shown in Fig. 1. A gene is reading frame of 2721 bases, coding 907 amino acids and determining the product of primary broadcast about 95 KD. The difference between the predicted and actual values associated with heavy glycosylation of the protein. 591 base (code 197 amino acids) are deleted as a result of splicing to avoid producing gp220. The apparent molecular weight of gp350/220 gene product may also vary depending on the type of measuring system, utilization of glycosylation sites and different types of cells, differences in post-translational processing or selective gene mutations. The measured values vary to produce different variants of non-splicing sites gp350/220 gene, but the term "homogeneous gp350 protein or protein" encompasses the gene products of non-splicing variants, optionally containing additional deletions or mutations, such as C-terminal deletions and/or transmembrane modifications are also described here. The term "gp220 protein" refers to alternative splanirovannim gp350/220 product with a molecular weight of about 220 KD. Splicing sites in the gp350/220 gene product were ODA is awns, based on other genes, mainly from eukaryotic organisms. Consensus sequences identified Mount, Nucleic Acids Res. 10: 459 (1982) in the study of splicing sites in other genes, have the form:

< / BR>
Stars at the base of the top to indicate the reason that appear in 100% of all sites of splicing (highly conservative). Position two reasons one reason or represent conservative position. The / character indicates the real site of splicing.

In the gp350/220 gene donor site splicing is after nucleotide 1051, and the acceptor site of splicing after nucleotide 2092, as can be seen from the DNA sequence (Biggin EMBO J. 3: 1083 (1984)) gene gp350/220. (Used here numbering and numbering in Fig. 1 corresponds to the numbering in Biggin). The website splicing is located in the corresponding portion of a gene in EBV strain type (donor splicing site after A1501and acceptor splicing site after G2029). The present invention encompasses compositions obtained using the splicing sites or And or strains or other EBV strains to retrieve a single species of mRNA of the gp350/220 gene. DNA sequence type And virus strain B95-8 was used in the examples, although t is thou strains of type a and b are identical. In the strain lacking amino acids 507-520 and 570-576. Strain type And use due to the fact that it contains all possible gp350 antigenic sites. Alternatively, you can use the EBV gp350/220 containing the strain-specific sequences in accordance with the existing guidance that will be produced EBV strain-specific homogeneous gp350 proteins with immunogenic properties specific to the strain, and therefore suitable for use in immunogenic and/or therapeutic compositions for the prevention or treatment of diseases associated with strain-specific EBV. Table 1 shows the wild-type nucleotide and amino acid sequences for donor and acceptor splicing sites.

To prevent RNA splicing of the gp350/220 gene, the sequence of nucleic acids gp350/220 gene introduce a mutation to replace the corresponding base pairs in the site of RNA splicing. To give dysfunction site splicing, preferably, at least one of the bases selected from two highly conservative basis, limiting the donor site or acceptor site, should be replaced by non-conservative bases, more predpochtnija. Other conservative base, at a distance of more than two bases from the site of splicing, it is also possible to replace the non-conservative foundations site splicing for further deterioration of the recognition site of splicing. As donor and acceptor sites can be modified to disrupt the mechanism of splicing. Preferably, both the donor and the acceptor contained at least one change in each one of the four highly conservative provisions of the reasons customers splicing, and more preferably, at least two changes in two of the four highly conservative provisions of the grounds of the site of splicing. If one of the sites of splicing may not be subjected to mutations because of the desire to preserve the wild type amino acid sequence, then it is preferable to introduce at least two mutations in another site of splicing.

Mutations in gp350/220 splicing sites can introduce changes in the amino acid sequence expressed later gp350 protein. It is preferable that such changes would be conservative substitutions of amino acids. Conservative substitutions in the amino acid sequence, in contrast nekonservativnykh of aminocyclo can be done as long until the change of bases (or modified bases) will not lead to appropriate changes in the invariant donor/acceptor substrates. For example, Gly may be replaced with Ser501in the splicing donor site, using any Gly-specific codon that is different from GGU (using GGU stores G nucleotide and will not lead to desirable replacement of the GT signal splicing). Similarly, the acceptor splicing site replacement of Gly698on A1A is a conservative change, but since all A1A codons begin with highly conservative G nucleotide, this may not lead to the desired replacement. Although Proline can also be change conservative amino acids, Proline should not be used to replace the wild-type amino acids, as this will lead to a modification of the tertiary structure of the protein, and this will mask one or more of the gp350 antigenic sites. Table 1 presents an acceptable conservative amino acid substitution in the wild-type sequences. At the bottom of table 1 presents an example of a mutation with conservative amino acid changes.

Although one aspect of the present invention includes nonspecific option Dr/220, may be desirable and more who are proteins or free in solution, or associated with the membrane, but not integrated with the membrane), for example, to avoid problems of cellular toxicity that occurs when the expression of full length Dr as an integral membrane protein that connects with the membrane area (also known as transmembrane segment) Dr modify due to the deletion of all or part of its coding DNA sequence. Connecting with a membrane area Dr/220 includes 861 amino acids (methionine) 881 (alanine). Cm. Beisel, J. Virology 54: 665 (1985). Preferably, at least 8 amino acids in the transmembrane region had been subjected to deletions, more preferably been subjected to the deletion of 12 amino acids, and most preferably been subjected to deletions from 12 to 18 amino acids. Accordingly, in another aspect of the present invention proposed a non-splicing variants of gp350/220 DNA and/or homogeneous gp350 proteins, which additionally contain at least one deletion in the transmembrane site gp350/220 DNA and/or homogeneous gp350 protein, which leads to the expression of a soluble, homogeneous gp350 protein.

In addition to deletions of all or part of the transmembrane domain of the non-splicing gp350/220 option, you can also moderncommando domain including amino acids 881-907, in accordance with the present invention. Thus, in another aspect of the present invention includes a non-splicing variants of gp350/220 DNA and/or homogeneous protein, optionally modified due to the deletion of all or part of the coding DNA and/or amino acid sequence containing transmembrane plot gp350/220 and even additionally modified due to the deletion of the rest of the C-terminal DNA and/or amino acid sequence of gp350/220.

Accordingly, another aspect of the present invention includes a non-splicing variants of DNA sequences encoding homogeneous gp350 proteins of the present invention. Such DNA sequences include DNA sequence shown in Fig. 1, coding for amino acids 1-907, and further include nucleotide substitutions, which were discussed, removal of donor and acceptor Saitov splicing. Such DNA sequences include optional truncated DNA sequence in which the nucleotides encoding the whole or part of the transmembrane domain and the C-end, including amino acids 861-907 undergo deletions, and options deletions, in which the nucleotides encoding the C the activity of the present invention, encoding homogeneous gp350 proteins, may also include DNA capable of hybridization under conditions of appropriate stringency, or which may be capable of hybridization under such conditions, but without destruction of the genetic code to the DNA sequence shown in Fig. 1. Accordingly, the DNA sequence of the present invention may contain modifications in non-coding sequences, signal sequences or coding sequences, on the basis of allelic variants or intentional modifications.

These non-splicing variants of gp350/220 DNA sequences, as disclosed here, can be constructed using well known in the art methods. Modified DNA sequence of the present invention can Express recombinante using known methods to obtain a homogeneous gp350 proteins of the present invention. Such recombinant proteins can be extracted and included in pharmaceutical compositions for prophylactic treatment and prevention of diseases associated with EBV.

Non-splicing variants of gp350/220 DNA of the present invention can Express recombinante in decomp is. Suitable cells known and available to the experts, include (but are not limited to) such yeast cells are Saccharomyces cerevisiae, these bacterial cells are E. coli and Bacillus subtilis, and such mammalian cells, as GH3, Cho, NSO, MDCK and C-127 cells. The vectors that are used with these types of cells, chosen on the basis of their compatibility with the type of cells used by vector control. Cells and vectors that provide for the expression of secreted products gp350/220 gene, are preferred. For example, E. coli transformed using derivatives of pBR322, which was modified using conventional techniques such that it contained the DNA sequence for the expression of a target protein, in this case, the non-splicing variant sequence EBV gp350, whether or not containing sequences encoding the C-ends and/or the piece that connects with the membrane. pBR322 contains genes for resistance to ampicillin and tetracycline, which can be used as markers. Cm. Bolivar, Gene 2: 95 (1977). Commonly used sequences control expression, i.e. promoters to initiate transcription and optional operator or enhancer include beta-lactamase) and the lambda-derived PL promoter and N-gene ribosome binding site (see Shimatake, Nature 292: 128 (1981)). However, you can use any available promoter system or control system for the expression, unless it is compatible with prokaryotic cells of the host. Other examples of cell owners, plasmids and expression vectors are disclosed in U.S. patent issued Itakura (1982) under 4356270, 4431739 issued by Riggs (1984) and 440859 issued by Rutter (1984).

As host cells can also be used based on insect cells using the expression of insect cells. In the case of expression in insect cells, usually the components of the expression system include vector transfer, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for integration of the heterologous gene or genes that need to Express; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the vector transfer (this helps to ensure homologous recombination of heterologous gene into the baculovirus genome); and the corresponding cells of the insect host and growth environment.

Currently, the most commonly used vector transfer for integration of foreign genes into AcNPV is RS. It was also designed m the start codon from ATG to ATT, and which introduces a BamHI site cloning of 32 base pairs in the forward direction from the ATT; see Luckow and Summers, Virology 17: 31 (1989).

Plasmid usually also contains the polyhedrin polyadenylation signal (Miller et. al (1988) Ann. Rev. Environ. , 42: 177) and the prokaryotic gene for resistance to ampicillin (amp) and the source of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Baculovirus promoter is any DNA sequence capable of binding baculovirus RNA polymerase and initiate in the forward direction (5' to 3') transcription of the coding sequence (i.e. structural gene) into mRNA. The promoter must contain the site of initiation of transcription, which is usually located proximally relative to the 5' end of the coding sequence. This site of initiation of transcription typically includes the site of binding RNA polymerase and initiating transcription. Baculovirus vector transfer may also contain a second domain, called enhancers, which, if present, is usually located distal of the structural gene. The expression can be either adjustable or constructive. For technology expression in insect cells, see EP patent publication 155476.

Of course, you can use eukaryotic cells from multicellular organisms as host cells for the expression of genes encoding the proteins or polypeptides of interest. Suitable cell lines hosts include VERO and HeLa cells, cells of the Chinese hamster ovary (Cho). The expression vectors compatible with such cells, are also available and usually include a promoter and a sequence controlling expression, such as for example, the early and late promoters from SV40 (see Fiers, Nature 273: 113 (1978)) and the promoters of the virus polyoma, adenovirus 2, virus bovine papilloma or avian sarcoma. Examples of host cells, promoters, breeding markers and methods also are disclosed in U.S. patents: 5122469 issued by Mather (1992), 4399216 issued by Axel (1983), 4634665 issued by Axel (1987), 4713339 issued by Levinson (1987), 4656134 issued Ringold (1987), 4822736 issued Kell is hartie techniques specific cells, such as CaCl2treatment for prokaryotes, as referred to in article Cohen Proc. Natl. Acad. Sci 69: 2110 (1972) and Saro4deposition to mammalian cells, as disclosed in Graham, Virology 52: 546 (1978). Transformation of yeast can be performed as described in Hsiao, Proc. Natl. Acad. Sci 76: 3829 (1979) or as described by Klebe, Gene 25: 333 (1983).

Construction of suitable vectors containing non-splicing variants gp350 sequence (with additional modifications (or without them), opened here and leading to deletion of the C-end and/or binding with membrane area), carried out using conventional methods of ligation and restriction, well now well-known specialists. Site-specific DNA cleavage is carried out, processing appropriate restriction enzyme (enzymes) under standard conditions, in particular in those which are usually specified by the manufacturers of restriction enzymes. For separation by size of the split fragments can be used electrophoresis on polyacrylamide or agarose gel using standard techniques, and to blunt the ends of the fragments, by processing the fragment maple E. Li polymerase 1 in the presence of four deoxynucleotide triphosphate is given, using, for example, diethylphosphoramidite method known in the art. Cm. U.S. patent 4415732 (1983). Ligation can be carried out using T4 DNA ligase under standard conditions and temperatures, and the correctness of the ligating confirm transforming E. coli or COS cells ligiously mixture. Successful transformants are selected by resistance to ampicillin, tetracycline or other antibiotics, or using other well-known specialists markers.

Such recombinant DNA techniques are fully disclosed in the literature. Cm. : Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED, (1989); DNA CLONING, Vol 1 and 11 (DN Glover ed 1985); OLIGONUCLEOTIDE SYNTHESIS (MG Gait ed 1984); NUCLEIC ACID HYBRIDIZATION (Hames BD ed 1984); REDUCED AND TRANSLATION (Hames BD 1984); ANIMAL CELL CULTURE (Freshney RI ed 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (JH Miller ed 1987 Cold Spring Harbor Laboratory); Scopes PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, 2nd ed, (1987 Springer-Verlag NY) and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY Vols 1-1V (DM Weired 1986).

All of these publications are included here with links.

Accordingly, in this aspect of the invention includes vectors containing non-splicing variants of gp350/220 DNA sequences and cell hosts, and further includes a method of obtaining a non-splicing variant Dr/220 protein due kultivirovalos, operatively associated with the sequence controlling the expression in culture conditions that provide for expression of a homogeneous gp350 protein.

Downregulation of homogeneous gp350 isolated from the cells and components of the culture medium using such conventional methods of purification of glycoproteins, as ultrafiltration, electrophoresis in free flow, gel filtration chromatography, affinity chromatography, SDS-PAGE, differential NH4SO4deposition, lachinova columns, ion exchange columns and a hydrophobic column (but not limited to! ), is well known to specialists. A small number of drugs gp350 most easily isolated using SDS-PAGE or lachinova affinity column and the small amounts of drugs for use in experiments on vaccination, or experiments on immune response is most easily cleaned using liquid chromatography. For large-scale production of commercially significant quantities gp350 for use in vaccine compositions of the preferred combination of ultrafiltration, gel filtration, ion exchange and chromatography hydrophobic interactions.

Purified homogeneous gp350 proteins infusion is ecene States, associated with EBV, and diseases such as infectious mononucleosis, Burkett lymphoma and carcinoma of the nasopharynx. Such pharmaceutical compositions include immunogene-inducing amount of one or more of the homogeneous gp350 proteins of the present invention in a mixture with a pharmaceutically acceptable carrier, for example, such a system representing adjuvant/antigen, as alum. You can use these other systems, representing adjuvant/antigen, as MF59 (Chiron Corp), QS-21 (Cambridge Biotech Corp), 3-DMPL (3-Deacyl-monophosphoryl lipid A 3-Deacyl-Monophosphoryl Lipid A), (Ribi-Mipo-Chem Research, Inc. ), clinical purity adjuvant Freund (IFA), photogenie liposomes, water-soluble polymers or Iscoms (immunostimulating complexes). Other examples of pharmaceutically acceptable carriers or solutions include aluminum hydroxide, saline or phosphate superyoung saline. These compositions can be entered systemically, preferably subcutaneously or intramuscularly in the form of acceptable solutions for subcutaneous or intramuscular injection. Inoculation can also be accomplished by causing scratches on the surface or by inoculation into the body cavity. The preparation of such solutions with through the pod into account such factors as physical condition, weight, sex, diet, the severity of the condition, time of administration and other clinical factors that may influence the effect of the drug. Examples of doses represent the range from about 1 µg to about 1000 µg of protein.

In the practice of the method of treatment of the present invention immunological-inducing effective amount of a homogeneous gp350 protein is administered to a patient in need of therapeutic or prophylactic treatment. Immunogene-inducing effective amount of the composition of the present invention is a value in the range from about 1 μg to about 1 mg per injected dose. The amount of injected doses may vary depending on the above factors. Hereinafter the invention is described in the following examples, without limiting its scope.

Example 1

The deletion of gp350/220 transmembrane segment and the transmembrane region up To-end to create pDTM and pSTOP

Gene gp350/220 of the EBV B95-stamma (Miller et. al, 1972) is available in the BamHI library as an open reading frame called BLLF1 (Baer, Nature, 310: 207, 1984). To create the desired design (see shown in Fig. 2B chart) gp350/220 gene clone in two parts: (1) BLSH1, 2.3 kb HindIII/BfaI 3' fragment and 2) BLSH2, 337bp BanI/ HindIII 5' fragment of plasticheskih and/or transmembrane-coding domains. As BfaI site is located at the 5' end of the segment encoding gp350 transmembrane (TM) domain, it is used to construct deletions TM domain and deletion of the TM domain in conjunction with deletions adjacent To-end. Using BfaI, it is possible to carry out deletions, leaving only two amino acids of the TM plot (table 2).

1. The design of pDTM pSTGl and pSTG3.

Plasmid pDTM consists of a sequence of gp350/220 nucleic acid that lacks the entire TM encoding section. This design is created using two Staging vector pSTGl and pSTG3. PCR product of 450 bp, SYCT, which introduces a BfaI site at the 3' end of the TM area, get, using BLLF1 clone micheneau sequence (Fig. 2).

As PCR primers using the following (see primer 1 and primer 2 in the end of the description).

BfaI site primer 1 used for cloning BfaI/ XmaI fragment SCYT in pSTGl. Primer 2 corresponds to the area outside Dr/220 open reading frame with the 3' side of the gene. SCYT PCR fragment is cut out due to the BfaI and XmaI to obtain fragment 136 base pairs that clone in pMT11 vector (Spaete and Mocarski, 1985) together with a second fragment, BLSH1 HindIII/BfaI fragment, to create pSTGl. Sequencing by BfaI site shows that was on the LF1 fragment, BLSH2, clone in pMT11 to create pSTG3. BanI/XbaI oligonucleotide linker of 16 base pairs outside Dr/220 gene coding sequences used for cloning BLSH2 BanI/Hindlll fragment in pSTG3. 2,4 HindIII/XmaI pSTGl fragment of clone in REE vector (Celltech, England) together with 0.3 XbaI/HindIII pSTG3 fragment to complete pDTM design.

2. Design pSTOP, using vectors pSTG2 and pSTG3.

Plasmid pSTOP contains gp350/220 gene lacking the TM site and the C-terminal cytoplasmically area adjacent to the TM site. To create this design creates BfaI/EcoRI oligonucleotide linker of 16 base pairs with stop codons (underlined) in the three frames following the BfaI sticky end, as shown here:

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5' dangling end (TA) the upper sequence is the sticky end for BfaI restriction site, and 5' overhanging end (TTAA) the lower sequence is EcoRI sticky end. This 16b the linker used to clone the fragment BLSH1 HindIII/BfaI in RMT to create pSTG2. the 2.3 kb pSTG2 HindIII/EcoRI fragment and pSTG3 0,3 kb XbaI/HindIII fragment of clone in REE to create pSTOP.

3. Comparison of wild-type, pDTM and pSTOP sequences on the TM site.

The oligonucleotide posledovatelno sequence presented below in table 2. Arrows indicate the beginning and the end of the wild-type transmembrane domain (TM). Only two amino acids from the transmembrane domain are stored in pDTM and pSTOP Mets861and Leu862(see also Fig. 1). It should be noted that the stop codon immediately follows Leu862in pSTOP. In pDTM position deletirovanie membrane area marked as "TM". (Table 2 shows the native amino acids).

Example 2

The removal of the gp350/220 gene donor and acceptor splicing sites to create pMDTM and pMSTOP.

To obtain a homogeneous production Dr protein substitute highly conservative and the conservative base of the splicing site Dr/220 gene. Four bases substituted at the splicing donor site, including highly conservative GT pair, which is 100% of all splicing sites. Two conservative Foundation donor site, AA, replace the GT. Two highly conservative (invariant) of the Foundation donor splicing site replaced with GT on CA. The acceptor splicing site, only highly conservative base of the splicing site substitute for the conservation of amino acid sequence. The second conservative base acceptor playinhobo the splicing sites gp350/220 gene.

The change of bases due to mutagenesis-based oligonucleotides observed in the mutant sequence stars. The actual splicing sites indicated by arrows, and presents the encoded amino acids. None of these amino acid sequences does not change the result of nucleotide substitution.

Nucleotide substitution in the wild-type gp350/220 DNA sequences donor splicing site and the acceptor splicing site carried out using mutagenesis, mediate the oligonucleotides. For the implementation of the mutations using a modified phage vector MTS according to the method of Zoller, M. E. and Smith, M. (1983) Methods of Enzymol. 100: 468. BamHI/XhoI fragments Dr/220 nucleotide sequence of the clone in polylinker plasmids MTS using s718 and BamHI restriction sites on polylinker, combined with 19 bp oligonucleotide linker containing the Asp718 and XhoI sticky ends. For mutagenesis using M13DTM and M13STOP of example 1 (Fig. 2B).

For use in mutagenesis create two 42-dimensional oligonucleotide, Gdong and Laccato. Each design so that it was complementary to the gp350/220 gene sequences tsentrirovannoi or donor, either p is any reason, representing the target mutation. Oligonucleotides after mutagenesis have the following form (see the end of the oligonucleotides after mutagenesis).

The sequence of the oligonucleotides after mutagenesis marked as "primer", whereas DNA sequence linking splicing sites Dr/220 gene marked EBV". The Foundation, which changed as a result of mutagenesis are marked with asterisks. The dashed line indicates the position of splicing.

Oligonucleotide Gdong and Laccato hydrolyzing to obtain single-stranded M13 clones DTM and M13-STOP. Holoenzyme TANK polymerase is used to produce double-stranded DNA, and this double-stranded DNA transform E. coli. Using vector MTS, any of the clones that contain the target mutation can be identified by the color change from white to blue in the presence of X-gal and sociopragmatic. Blue plaques are selected and grown, and the DNA sequence for connections splicing is used for final identification of mutant clones marked as M13-MDTM and M13-MSTOP.

After identification of clones containing the target mutation, fragments BamHI/XhoI cut out from M13-MDTM and M13-MSTOP and are ligated back into pDTM and pSTOP main circuit DL is leasingowych options gp350/220 DNA sequences, as indicated in example 3.

Example 3

Expression of gp350 in Cho cells.

1. Transfection of gp350/220 gene construct.

One way of obtaining high yields homogeneous gp350 protein of the present invention from mammalian cells involves the construction of cells containing multiple copies of the heterologous gp350 sequence. Heterologous DNA sequence operatively linked to amplificare marker, in this example, the glutamine synthase gene, for which cells you can amplify using methanesulfonamide. Vectors pMDTM and pMSTOP obtained in example 2, transfection in Cho cells, as will be indicated hereinafter by way Crockett, Bio/Technology 8: 662 (1990) and as specified in Celltech Instruction Manual for the system, amplification of gene of glutamine synthase (1992).

Cho-K1 cells (ATSC CCR61) support in not containing glutamine of EMEM (Eagles minimal maintenance medium), supplemented with 10% serum, fetal calf, 100 u/ml penicillin, 100 mg/ml streptomycin, MEM (modified atmosphere Eaglets) nonessential amino acids and 1 mm sodium pyruvate (all obtained from JRH Biosciences). The medium is supplemented with 60 mg/ml glutamic acid, 60 mg/ml asparagine, 7 mg/ml adenosine, 7 mg/ml huangshanshi (and, in case of deviation from this recipe are instructions).

The day before transfection 10-cm Cup seeded 3x106Cho-K1 cells. On the day of transfection, the cells washed with 10 ml of medium containing no serum (for each Cup). Plasmid DNA (pMDTM and pMSTOP plasmids) contribute through Saro4deposition using conventional techniques. 10 sediment µg of each plasmid DNA incubated with Cho-K1 cells plus 2 ml containing no serum medium at 37oWith over 4.5 hours. Perform a triple experiment for each of the four plasmid transpency. The cells are then shaken for 1.5 minutes with 15% glycerol in saline solution, bateriafina HEPES. After washing does not contain serum environment, it is substituted for containing serum medium, and the cells incubated for 24 hours.

The next day the medium was replaced with medium containing 10% cialisbuynow bovine serum (JRH Biosciences) and amplified by adding 25 μm methanesulfonamide (Sigma). The medium containing methanesulfonamide, updated every 3-5 days before until amplificatoare clones are not large enough, so that they can be collected, i.e. after about 13-14 days. Clones gather, coscribe the cells in medium without methanesulfonamide. After 1-2 days the medium was replaced by medium with addition of 25 μm methanesulfonamide. After 4 days, collect the supernatant and analyze the content of the target protein in an ELISA assay. Cho cells transfection REE control vector alone (which does not contain EBV sequences) and 24 of the clone Cho-REE also collected and transferred to plates for controls. (Control clones identified on the basis of survival in methanesulfonamide).

2. ELISA analysis.

After transfection 241 clone CHO-pMDTM and 158 clones SNO-pMSTOP gather and grow. The supernatant of these clones are tested for the purpose of obtaining gp350 protein. 96-yacheechnye plate cover afinno-purified rabbit anti-gp350/220 antibody (antibodies MDP1, a gift from Andrew Morgan), diluted 1: 2000 in 50 mm atrebatum buffer, pH 9. Plates are incubated for 3-4 hours and three times washed with PBS + 0.05% tween-20 using Nunc Immune Washer. After the dried blots, plate block, incubare with 2% BSA in PBS + 0.01% of Thimerosal at 37oWith in half an hour, and washed again. The supernatant liquid transfection cells and control cells are placed in the cells and incubated for 2 hours at 37oC. Then the plates national) at 1 mg/ml diluted in PBS wash buffer at 37oC for 1 hour. After washing, plates are incubated with the second antibody, fragments of goat F(ab)2conjugated with horseradish peroxidase directed against mouse immunoglobulins (Ig adsorbed person; Biosource International), and 0.7 μg/ml in PBS + 0,05% BSA and 0.01% Thimerosal at 37oC for 1 hour. Plates are washed and are using ABTS (Pierce Chemicals) dissolved in Stable Peroxide Substrate Buffer (Pierce Chemicals) for 0.5 hours at room temperature. The reaction is stopped with 1% SDS and the plate read at 405 and 650 nm using a card reader Molecular Devices Vmax ELISA. 24 pMDTM and 18 pMSTOP colonies were tested as positive in relation to secretio of gp350. The colony, which gave the most intense ELISA signals were transferred to plates with 24 cells for reproduction and further analysis by Western blot testing and analysis radioimmunoassay.

3. The Western blot testing and analysis radioimmunoassay.

In the initial skanirovaniya supernatant tissue cultures of pMDTM of transpency analyze activity in Western blot testing. The supernatant of Cho cells purified on 5% SDS-PAGE gels, transferred to nitrocellulose after night and probe anti-gp350 antibodies. Seven pMDTM clones okazyval the Western blot testing, further testing using radioimmunoassay on the subject of presence gp220. The selected transformed with pMDTM cells, REE control and GH319 control cells (described later) are grown overnight in plates with 6 cells so that they were approximately three quarters of confluent on the day of the experiment. Each cell contains approximately 5106cells. For the introduction of a label medium from each cell removed and replaced with 0.7 ml MEM containing no methionine (10% serum fetal calf) + 100 mccoury35S-mation. Cells incubated for 5.5 hours at 37oC, and then treated in microcentrifuge at a speed of 4000 rpm for 5 minutes. Homogeneous gp350 protein in the supernatant immunoassays by adding 10 μl of Sepharose-Protein A (Sigma) in 50% suspension and 20 μl of monoclonal anti-gp350/220 (antibodies SM, 100 μg/ml, Biodesign International) overnight with shaking at 4oC. the mixture is Then precipitated at 2000 rpm for 2 minutes at room temperature in microcentrifuge, washed four times with small volumes superyoung phosphate saline solution. After the last wash all the liquid removed from the sludge and replaced with 50 μl protein gel sample buffer. Samples containing osujdenni the supernatant fluids, tissue cultures, mixed 1: 1 with protein exemplary buffer. The gel is dried and autoradiographed on Hyperfilm-Max (Amersham).

In Fig. 3 presents the results autoradiographical research results SDS-PAGE analysis radioimmunoguided designs. As a control using the cell line GH319 (gift Elliot Keiff; Whang et al. , 1987).

Cells GH319 secrete truncated form of the protein gp350/220, does not contain the transmembrane and C-terminal tsitoplazmaticheskogo domain. For negative control cells SNO transfection only one vector REE and selectyou using methanesulfonamide in parallel with pMDTM by transfection. In Fig. 3 supernatant (S) presented in odd-numbered bands, alternating with immunostaining ("IP"), presented in the even-numbered bands. In the control lane 2, deposition of GH319 control cells results in two intense protein bands at approximately 220 and 350 CD that demonstrates the production of truncated splicing variants gp350 and gp220 proteins in a ratio of about 1: 1. As expected, these immunosurgery band concentrated compared to the supernatant fluids radiometric tissue cultures (not subjected to immunoassay samples) in which contains any of the gp350/220 designs.

SDS-PAGE analysis of immunosurgery of the supernatant liquids pMDTM clones in lanes 6, 8 and 10 leads to the emergence of a single intense band of approximately 350 KD, similar to the more high molecular weight samples in GH319 control strip 2.

However, in contrast GH319 control band more intense band of approximately 220 KD absent in lanes 6, 8 and 10, although in the band 8 is very weak strip is displaced in area of slightly lower molecular weights. This may be a decomposition product, precipitated together cellular product or a small number gp220 protein resulting from incorrect broadcast or acts of mutations that return excluded donor and acceptor splicing sites in the native nucleotide or amino acid sequence. Intense single band at about 350 KD were detected in five other tested MDTM the replicate (data not shown).

It is unlikely that the complete absence of bands at 220 KD in lanes 6 and 10 is associated with inefficient deposition from MDTM supernatant liquids, as in35S-labeled GH319 control strip (2) band at 220 KD clearly visualized. In addition, the complement is the CIO to receive two intense bands at 350 and 220 KD. Therefore, these results demonstrate the fact that the deletion sites of splicing leads to the production of gp350 protein without the simultaneous production of gp220 protein.

This homogeneous gp350 protein, expressed in Cho cell lines, or in other mammalian cell lines, it is possible to get even larger scale, and homogeneous gp350 protein can be extracted and cleaned from conditioned medium from cell lines using methods known in the art, including techniques such as lectin-affinity chromatography, HPLC with reversed-phase, high-speed HPLC, gel filtration, and so on . Cm. David, J. Mipo Methods 108: 231 (1988) and Madej, Vaccine 10: 777 (1992).

4. Maserbating pMDTM protein.

In this experiment, using Maserbating shown that cells MDTM-1 produce gp350 RNA, but not gp220 RNA, confirming, thus, on another level, that mutation splicing site prevent the production of gp220.

DNA probes complementary gp350, were obtained from pDTM, see example 1. The matrix probe gp350/220, HR, isolated in the form of 464 bp XhoI/ > PST pDTM fragment. HR recognizes as gp350 and gp220. To obtain gp350-specific probe AN537, pDTM cut through NcoI and NdeI, there are two overlapping fragment of about 580 bp, II fragment inside gp350/220 splicing site. AN537 specific area, splanirovano of gp220 and, thus, gp350-specific message. DNA probes mark32P-dCTP nick-translation (Amersham), using DNA fragments HR and AN537.

Whole cell RNA receive almost in accordance with the method Chomczyunski and Sacchi, Anal. Biochem. , 162: 156-59 (1987). The two T-250 flasks each SNO-REE (as a negative control line, see example 1), SNO-MDTM and CHO-DTM-7 cells, 90% confluent, sucked off and the cells are subjected to lysis and scraped in denaturing buffer (10 ml guanidinoacetate, 25 mm sodium citrate, pH 7,05 sarkosyl, 100 mm 2-mercaptoethanol). Every 10 ml of lysate supplemented with 1 ml of 2M sodium acetate, pH 4, 10 ml of saturated phenol, pH 4.5 and 2 ml of a mixture of chloroform/soamiely alcohol; the resulting lysate is incubated on ice for 15 minutes, rotate at 10,000 g for 20 minutes at 4oAnd the upper aqueous phase is removed. RNA is precipitated from the aqueous phase by adding one volume of isopropanol at 20oC for 1 hour, precipitated at 4oWith, again suspended in denaturing buffer and again precipitated. The RNA precipitate was washed with 1X 70% ethanol, dried in a Speed-Vac and again suspended in DEPC-treated water.

DTM-7 and MDTM-1 total cellular RNA denatured at 65oWith over 15 m of the cellulose due to capillary interactions, then probe labeled HR and AN537. DNA zone denatured by boiling for 5 minutes, hybridized in 5X SSPE at 65%during the night and the nitrocellulose is washed under conditions of high stringency. The autoradiograph carried out using Bio-Rad phosphoimager.

Maserbating full cellular RNA from CHO-MDTM cells demonstrates the efficiency of splicing mutations to prevent the production of gp220-specific RNA. gp350-specific probe, AN537, associated with only one type of RNA in MDTM-1 and DTM-7 cells (Fig. 4, lanes 1 and 2), as expected for Dr-350-specific probe. Probe HR, which is specific for gp350 and gp220 RNA recognizes two types in the DTM-7 and some more high molecular weight band at the MDTM-1 (Fig. 4, lanes 4 and 3), as expected if splicing mutations prevent the production of gp220 message in MDTM-1. Even if MDTM-1 band is overloaded for gp350-specific RNA was not observed visible gp220 RNA. The reason for the differences in apparent mobility gp350 message in MDTM-1 compared with the DTM-7 stripe unknown. Types MDTM-1 overloaded compared to the DTM-7, which may affect the migration in the gel. In addition, gp350 message comes near to intensive ribosomal RNA band on the gel, which may affect the apparent m moznosti to assume, this signal is gp350 mRNA. Thus, mutations in the donor and acceptor splicing sites effective to prevent the production of gp220 mRNA according to Maserbating. This result is further confirmed by radioimmunoassay using monoclonal antibodies specific for gp350/220, as well as by Western blot testing MDTM-1 and DTM-7 supernatant liquids.

Example 4

Testing homogeneous gp350 proteins in the immunogenic activity.

Purified homogeneous gp350 proteins injected into appropriate carriers for injection and administered to mice as follows.

2 adjuvant-media concentrate is prepared by mixing Pluronic L121 and squalane 0.4% (vol/vol) tween 80 in bateriafina phosphate saline solution (Thr1) MDP in accordance with the way David, J. Immunol. Methods 108: 231 (1988), and Allison, J. Immunol. Methods 95: 157 (1986).

The composition for injection is prepared by adding equal volumes of protein and adjuvant carrier on the day of introduction. Protein content should be in the range from 5 μg to 50 μg per dose.

Mice of the strain BALB/c immunized three 0.1 ml intramuscular injection at 0, 21 and 42 day. Blood from pre-immunogenic consistently on day 10 PT in the ELISA according to the method of example 3. EBV neutralizing antibodies in serum count on their ability to inhibit the transformation of b-lymphocytes in the blood of the spinal cord of the fetus in EBV in vitro according to the method Moss, J. Virol. 17: 233 (1972) and De Schryver, Int. J. Cancer 13: 353 (1974).

Accordingly, new Zealand white rabbits inoculant intramuscularly five doses of protein emulsified in the previously mentioned adiuvante 0, 21, 42, 63 and 84 days. The dose should be in the range of from about 5 μg to 50 μg per inoculation. Whey receive two weeks after the last dose and tested for titers of antibodies to antigen for cross-reactive antibodies to the virus gp350/220 from B95-8 cells and for in vitro EBV-neutralizing activity by methods Emini, Virology 166: 387 (1988).

Because of the ability of EBV gp350/220 protein to induce protective immunity against EBV infection in animal models have already been installed, see Epstein, Clin. Exp. Immunol 63: 485 (1986), we expect similar positive results from the introduction of the composition homogeneous gp350 protein.

Disclosure all here publications included here by reference. A detailed description is provided only for purposes of clarity and understanding, and is by no means limit the invention, which includes all modifications, kotoraya expression homogeneous gp350 protein.

2. The DNA sequence under item 1, characterized in that it contains a nucleotide sequence in which at least one native nucleotide that encodes a serine at position 501 in Fig. 1, replaced negativnym a nucleotide in which at least one native nucleotide encoding glycine at position 698, replaced negativnym the nucleotide.

3. The DNA sequence under item 1, encoding a homogeneous gp350 protein, which is further characterized by amino acid sequence selected from the group consisting of a) amino acids 19-862 in Fig. 1, b) amino acids 1-862 in Fig. 1, b) amino acids 19-862 and amino acids 882-907 in Fig. 1, d) amino acids 1-862 and amino acids 882-907 in Fig. 1, d) amino acids in Fig. 1, where the nucleotides encoding at least 8 amino acids in the transmembrane site has been deleted.

4. A vector containing the DNA sequence described in any of paragraphs. 1-3.

5. A method of obtaining a homogeneous gp350 protein, comprising culturing host cells, transformirovannykh a DNA sequence according to any one of paragraphs. 1-3, operatively associated with controlling the expression of a sequence capable of controlling the replication and expression of the specified ASS="ptx2">

6. Homogeneous gp350 protein, donor and acceptor splicing sites which are encoded by one or more oligonucleotides in which native nucleotides (nucleotides) substituted (replaced) replacement negativnym the nucleotides (nucleotides) with preservation of the amino acid sequences of these donor and acceptor splicing sites, obtained by the method described in paragraph 5.

7. Pharmaceutical composition for prophylactic treatment of EBV-associated disease or condition, including homogeneous gp350 protein, as described in paragraph 6, in a mixture with a pharmaceutically acceptable carrier.

8. Homogeneous gp350 protein used to obtain the pharmaceutical composition according to p. 8.

 

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