Live attenuated pasteurella multocida bacterium, based vaccine, method for making vaccine and using such bacterium for making vaccine for animal or human protection
SUBSTANCE: live attenuated Pasteurella multocida bacterium is modified by introduing a mutation into the gene Orf-15. The mutation represents insertion and/or deletion in the gene Orf-15. As a result of modification, the bacterium is not able to express the functional protein Orf-15 that leads to its attenuation. Also, disclosed is the use of such bacterium for preparing a related vaccine for human or animal protection against Pasteurella multocida bacteria infection or pathogenic effects of the infection.
EFFECT: increased efficacy of applying the composition.
16 cl, 1 dwg, 3 tbl, 6 ex
The present invention relates to live attenuated bacteria speciesPasteurella multocidato live attenuated vaccines containing these live attenuated bacteria for use of these bacteria for the production of these vaccines, to methods of manufacture of such vaccines and diagnostic tests for detection of these bacteria.
Gram-negative bacteriumPasteurella multocidaknown as the causative agent of the disease in several animal species over a century. As you know,Pasteurella multocidaamong other things, causes the bird cholera in poultry, haemorrhagic septicaemia in cattle and atrophic rhinitis in pigs. In addition, its importance as a human pathogen is becoming more and more evident in the last 60 years.
There is only one kind ofPasteurella multocida. Subspecies does not exist. Nevertheless, it is possible to perform division on the basis of differences in capsular antigen and LPS-antigen. Five groupsPasteurella multocida, A-E, were determined on the basis of capsular antigens, and 16 somatic serotypes were determined on the basis of LPS-antigen. On pathogenicity or virulence is not affected by the group capsular antigen or LPS serotype strains. Group capsular antigen merely identifies the animal host for each particular strain.
The strains that cause Friday is whose cholera, relate mainly to the group of capsular antigen of A. there are two types of diseases: acute and chronic avian cholera. The symptoms of acute avian cholera are depression, ruffled feathers, fever, anorexia, mucus and shortness of breath. Often such damage, as petechial and chemotechnique hemorrhage, General passive hyperemia and accumulation of peritoneal and pericardial fluids. The symptoms of the disease in chronically infected birds usually associated with localized infections. There is often a swelling of the piercer, sinuses, periorbital subcutaneous tissues, joints of legs or wings. There is often exudative conjunctivitis and pharyngitis. Damage in chronically infected birds usually characterized by fibrinous-purulent exudate, focal necrosis and proliferation of connective tissue.
The strains that cause hemorrhagic septicemia in cattle and Asian Buffalo and pigs, sheep, goats, deer and camels belong mainly to the group of capsular antigen and E. Hemorrhagic septicemia is an acute disease characterized by rapid flow, edematous swelling in the head and throat of the sternum, swollen lymph nodes with hemorrhage and the presence of numerous the subserous petechial hemorrhages.
In pigsPasteurella multocidacauses atrophic rhinitis and pneumonia. These syndromes are caused mainly by strains of capsular types a and d were Also described cases of acute septicemia, which were caused by capsular type C. Clinical signs associated with atrophic rhinitis include sneezing, runny nose, shortening and twitching snout, pneumonia, and growth retardation. Pneumonia is observed mainly as a secondary infection, increasing the severity of the primary injury. Clinical signs include a dry, unproductive cough that becomes productive, and, in severe cases, fever.
In principle, when it comes to vaccination againstPasteurella multocidathere are two approaches to the protection against infectionPasteurella multocida: vaccination with killed vaccines (bacteriae) and vaccination with live attenuated vaccines. Bacteria economically attractive, because their production is cheap. However, they need to be injected, they often cause severe tissue reactions, high pressure antigenic stimulation can still cause outbreaks of disease in vaccinated bacteriae animals and, worst of all, they provide protection only against the homologous serotype. In contrast, vaccination with live attenuated vacci the OI provides a good cross-protection, not only against the homologous serotype, but also against heterologous serotypes. Thus, these vaccines, it seems, must be the vaccine of choice; however, there are two major disadvantages associated with the use of live vaccines: first, live attenuated vaccine strains ofPasteurella multocidaused at the present time is ill-defined nature of weakening their behavior is unknown. Thus, there is always a risk return them to a virulent state. And secondly, there were outbreaks of disease pasteurellosis, which could be attributed to the used live vaccine strains. A possible cause of these outbreaks could be the return of the used vaccine strain to a virulent state or insufficient attenuation.
Thus, it is obvious that there is a need for attenuated vaccinesPasteurella multocidathat are both effective and safe. These vaccine should provide protection against infectionPasteurella multocidaor from its effects, and at the same time, should behave as attenuated, with no tendency to revert to virulence.
The aim of the present invention is the development of a live attenuated strain ofPasteurella multocidathat meets the specified requirements.
To date, it has been unexpectedly determined that the new, still n the p unknown, genePasteurella multocida(hereinafter referred to as the genome Orf-15, can be omitted without disrupting the immune reactivity of the strain, at the same time, with a dramatic reduction in virulence of the bacteria. The presence of the gene in wild-typePasteurella multocidanot limited to the specific capsular antigen or somatic serotype. The gene is present in strains ofPasteurella multocidaregardless of the group capsular antigen or somatic serotype: it is found in all 16 somatic serotypes and all five groups capsular antigen.
Thus, a gene is a universal goal of attenuatio thePasteurella multocidathat for the first time allows to make safe vaccines against diseases associated withPasteurella multocida. This approach, therefore, equally suitable for the manufacture of, for example, vaccines that protect people from infectionPasteurella multocidaor its effects, vaccines, protecting poultry against avian cholera vaccine protects pigs from atrophic rhinitis, and vaccines to protect cattle and Asian Buffalo from hemorrhagic septicemia.
In addition, it was found that live attenuated strains ofPasteurella multocidadeprived of gene Orf-15, not only provide very good protection against homologous them serotype, but also a very good cross-protection against heterologous ser the types.
Thus, the first variant implementation of the present invention relates to live attenuated bacteriaPasteurella multocidathat is not able to Express functional protein Orf-15. The sequence of a new open reading frame of the gene Orf-15 is presented in SEQ ID NO:1 and protein Orf-15, which it encodes, is depicted in SEQ ID NO:2.
Functional protein Orf-15, it seems, is a protein Orf-15 capable of full virulence, as inPasteurella multocidawild-type.
Thus, the strain ofPasteurella multocidathat is defective in at least the specified capacity, is unable to Express a functional protein Orf-15. Any mutation in the gene Orf-15, such as insertion, substitution or deletion mutation, which leads to the reduction of virulence compared to the strain ofPasteurella multocidawild-type, considered as included in the scope of the present invention.
Reduced virulence of the strain, for the purposes of the present invention is determined in two ways. One definition reduce virulence refers to the level of protection against lethal infection: it is known that infection by a strain ofPasteurella multocidawild type turkeys in controlled conditions gives a mortality of over 50% (see, inter alia, the “Examples”section). The strain ofPasteurella multocidawith the mind is isenau virulence due to mutations of the gene Orf-15 according to the invention is a strain, which under the same conditions gives a mortality rate less than 10%.
Another definition reduce virulence refers to the level and severity of lesions after vaccination compared with sublethal infection by a strain ofPasteurella multocidawild-type. Thus, according to the second definition decrease the virulence of the strain ofPasteurella multocidahas a lower level of virulence, if he causes damage, which is less than 30% of the damage level caused by infection by a strain ofPasteurella multocidawild-type.
Thus, the strain ofPasteurella multocidait is considered within the scope of the present invention, if it has, for example, a mutation in the Orf-15 or an agent inhibiting the expression of the proteins encoded by the genome Orf-15 (see below), which lead to a decrease in virulence, according to at least one of the two definitions of reducing virulence above.
This mutation may be an insertion, a deletion, substitution or a combination of both, provided that the mutation leads to an inability to Express functional Orf-15. (Needless to mention that the silent mutation, such as the mutation of codon CTC in CTT, does not affect protein Orf-15 and, thus, leading to inability to Express functional protein Orf-15. Therefore, this mutation is not suitable for the purposes for the present invention.
Usually the mutation can be an insertion, substitution or deletion of one or more nucleotides. Especially the insertion or deletion of a sequence of nucleotides that is not divisible by three, leads to a shift of the reading frame, which, in turn, leads to the appearance of meaningless code. The result will be synthesized truncated protein Orf-15 which has a reduced functionality or even has no functionality.
There are many known methods of introducing mutations in the open reading frame. One possible way to obtain these mutations is the use of classical methods, such as processing of wild-type bacteria mutagenic agents such as analogs of the grounds, the processing of UV light or thermal processing. Selection of mutants Orf-15 will, however, be a task that requires quite time-consuming.
In addition, the nature of mutations induced by classical methods of obtaining mutations is unknown. This can be a point mutation in the gene Orf-15, which eventually can return to the wild type. In order to avoid the specified low risk, a good alternative may be the transposon mutagenesis. Obtaining mutants using transposon mutagenesis well izvestno specialists. This is a type of mutation performed on localized segment of a chromosome. This way you can get a stable, avirulent, immunogenic, transposon-mediated mutantsP.multocida. Transposon-mediated mutants are mutants that have the transposon inserted into the bacterial genome. "Transposon" is an element of DNA that can be inserted into DNA molecules by transposition, the process of homologous recombination, which does not require extensive gomologichnosti DNA sequence. Transposons typically include genes encoding enzymes of transposition, called model, which cut the DNA at the end of transposons and paste transposons in the DNA target. In addition, transposons usually contain marker genes that encode resistance to antibiotics that can be used for selection of mutants with transposon-insertion. Known transposons include Tn3, Tn5, TnphoA, Tn7, Tn9, Tn10 and their functional fragments (Mobile DNA, ed. D.E. Berg and M.N. Howe, ASM Press, 1989). Just as an example, the transposon Tn10 well known in the art and are described, among others, Lee (Lee, M.D., Henk, A.D., Veterinary Microbiology, 50, 1996, 143-1480). A mutant with a transposon insertion can be obtained by standard methods known in the art (Mobile DNA, ed. D.E. Berg and M.N. Howe, ASM Press, 1989). Selection on the subject transposon the mutations Orf-15 will be easier and will take less time due to the fact, what PCR using primers located in the 3'end and 5'end of the gene Orf-15, it will directly show is the transposon insertion in a gene Orf-15 or somewhere else.
Even more elegant possibility of introducing a mutation in the Orf-15, this time in the pre-determined plot, rather deliberately, than coincidence that provides recombinant DNA technology, more specifically, site-specific mutagenesis. This mutation may also be an insertion, a deletion, a substitution of one nucleotide by another, or a combination of both, with the only condition that the mutated gene no longer encodes a functional Orf-15. Such mutation can be, for example, be achieved by deletion of a number of base pairs. Even very small deletions, such as deletion of a single base pair, resulting in a shift in the reading frame, can be sufficient to make the Orf-15 non-functional. More preferably, if you delete a longer period, for example, 10, 50, or more base pairs. Even more preferably, the gene Orf-15 eliminate entirely.
Method of designing Orf-15-negative mutants by site-specific mutagenesis are well known standard techniques. They include, for example, to clone the gene Orf-15, the modification of the gene sequence of pose the CTV site-specific mutagenesis, cleavage with restriction enzymes, followed by religionem or PCR approaches, and the subsequent substitution of the gene Orf-15 wild-type and mutant gene (allelic allelic exchange or substitution). Standard techniques of recombinant DNA, such as the cloning of Orf-15 plasmid, splitting the gene with restriction enzymes, followed by treatment with endonuclease, religioasa and homologous recombination in strain-owner, well-known in the art and are described, among others, in Maniatis/Sambrook (Sambrook, J. et al. Molecular cloning: a laboratory manual. ISBN 0-87969-309-6). Site-specific mutations can be done byin vitrosite-specific mutagenesis, using a set of Transformer® from Clontech. PCR techniques are widely described (Dieffenbac H & Dreksler, PCR primers, a laboratory manual ISBN-087969 and ISBN 0-087969-447-5).
The most common methods of constructing the live attenuated bacteriumPasteurella multocidathat is not able to Express functional protein Orf-15, based, as explained above, mutations of the gene Orf-15. However, there is an alternative way to receive live attenuated bacteriaPasteurella multocidathat is not able to Express functional protein Orf-15. The specified alternate path refers to the interaction with the messenger RNA that encodes a protein Orf-15. Expression of the protein is a two-step process is, includes a generation step of mRNA Orf-15 via the transcription of DNA and the subsequent translation stage indicated mRNA, obtaining protein Orf-15. The presence of certain types of RNA, such as Orf-15-specific ds, Orf-15-specific short interfering RNA or Orf-15-specific antisense RNA, bacteria can interfere with mRNA for Orf-15 and thus block translation of mRNA Orf-15 protein Orf-15 in quantities comparable to those in wild-type bacteria. RNA, which is based on this mechanism, as the mechanism as such, is widely known as RNC. Thus, the presence of, for example, the Orf-15-specific siRNAs, ds or Orf-15-specific antisense RNA, will cause the same effect that a mutation in the Orf-15: this bacterium is not able to Express functional protein Orf-15.
The use of antisense RNA for gene silencing, known for several decades, and the use of RNC (for example, ds or siRNAs), which is widely used for about five years, are well-developed, well-known specialists techniques. Overview on this topic written Hannon, G.J., Nature 418:244-251 (2002) and Deni, A.M. and Hannon, G.J., Trends in Biochemical Sciences 28: 196-201 (2003). Other articles describing the use of siRNAs for gene silencing, written Bertrand, J.R. et al., in B.B.R.C. 296: 1000-1004 (2002) and Sorensen, D.R. et al., in J. Mol. Biol. 327: 761-76 (2003). Use RNC for silencing viruses in mammals has been proposed recently, and, among others, written the review Quan-Chu Wang et al. (World J. Gastroenterol. 9: 1657-1661 (2003)).
Generally speaking, a specialist will probably give a slight preference for the first approach to obtain mutations of the gene Orf-15. This is due to the fact that the mutation of the gene, as opposed to any method based on RNA interference, does not introduce any additional genetic material into the cell.
Thus, the preferred form of this variant implementation of the present invention relates to live attenuated bacteria, which are unable to Express a functional protein Orf-15 due to the mutation of the gene Orf-15.
Deletion or insertion, especially mutation outside reading frames, will have a pronounced effect on the functionality of the protein Orf-15.
Thus, in the preferred form of this variant implementation of the present invention the mutation comprises an insertion and/or deletion. In the most preferred form is omitted, the entire gene Orf-15 or at least its coding sequence.
Gene Orf-15 includes the coding sequence and promoter region and the site of binding to ribosomes. The promoter region includes at least a region that includes the nucleotide 22-71 SEQ ID NO1, while the binding site with ribosomes includes nucleotides 92-96. Thus, it is needless to mention that any mutation that makes inefficient promoter or binding site with ribosomes, and thus, reduces the expression or termination of the expression of the Orf-15, is also in the scope of the present invention.
Taking into account the large amount of vaccines that currently impose domestic and farm animals, it is clear that it is desirable combined introduction of several vaccines, at least for reasons to lower costs of vaccination. Thus, it is very attractive is the use of live attenuated bacteria as recombinant carrier for heterologous genes encoding antigens selected from other pathogenic microorganisms or viruses. The introduction of such a recombinant carrier has the advantage that at the same time acquired immunity against two or more diseases. Live attenuated bacterium according to the invention provide a very stable carriers for heterologous genes, due to their attenuated behavior.
Thus, even more preferred form of the present invention relates to live attenuated bacterium according to the invention, to ora carries a heterologous gene, encoding one or more antigens selected from the group of microorganisms or viruses pathogenic for humans and/or animals.
The use of gene Orf-15 as insertion region for heterologous gene has the additional advantage that at the same time gene Orf-15 is inactivated, and the newly introduced heterologous gene can be expressed (in interaction with the homologous bacterial genes). Thus, even more preferred form of the present invention relates to live attenuated bacteriaPasteurella multocidaaccording to the invention, which as a distinctive sign is heterologous gene insertional in the gene encoding Orf-15.
Heterologous gene can carry homologous promoter or any other promoter that is recognized by RNA polymerase,Pasteurella multocida. The native promoter of Orf-15 can also be used. Most simply this can be achieved by deletion of the coding sequence of the Orf-15 and its replacement by a selected heterologous gene.
The design of these recombinant carriers can implement a routine way, using standard techniques of molecular biology as described above.
In one preferred form of implementation of the present invention, the heterologous gene encodes one or b is more antigens, selected from the group of pathogens of pigs, consisting of a virus reproductive respiratory syndrome swine (PRRS)virus pseudoleskeella virus, swine influenza, swine parvovirus, a virus, infectious gastroenteritis, rotavirus, swine circovirus 1 or 2,Escherichia coli,Erysipelothrix rhusiopathiae,Bordetella bronchiseptica,Haemophilus parasuis,Mycoplasma hyopneumoniaeandStreptococcus suis.
In another preferred form of implementation of the present invention, the heterologous gene encodes one or more antigens selected from the group of pathogens of cattle, consisting of herpes virus bovine virus viral diarrhea in cattle, parainfluenza virus type 3, paramyxovirus cattle virus, FMD,Pasteurella haemolytica,Staphylococcus aureus,Escherichia coli,Staphylococcus uberis, respiratory syncytial virus, bovine,Theileria parva,Theileria annulata,Babesia bovis,Babesia bigemina,Babesia major,Trypanosoma species,Anaplasma marginale,Anaplasma centraleorNeospora caninum.
In another preferred form of implementation of the present invention, the heterologous gene encodes one or more antigens selected from the group of pathogens in poultry, consisting of avian pox virus, infectious bronchitis virus, infectious bursitis (Gumboro), virus bole is neither Marek, agent anemia chickens, avian reovirus,Mycoplasma gallisepticum, a virus of turkeys,Haemophilus paragallinarum(Coryza), poxvirus chickens, avian encephalomyelitis virus, duck plague virus, Newcastle disease virus, syndrome dropping eggs, virus, infectious laryngotracheitis, herpes virus of turkeys,Eimeria species,Ornithobacterium rhinotracheale,Mycoplasma synoviae,Clostridium perfringens,Salmonella speciesandE. coli.
Another attractive possibility is the insertion, preferably, the gene Orf-15, the gene encoding a protein involved in triggering the immune system, such as a cytokine, interleukin or interferon, or other gene involved in the regulation of the immune system.
Due to their unexpected attenuated, immunogenic but the characterin vivothe bacteria according to the invention are very suitable as the basis for live attenuated vaccines.
Thus, another variant of implementation of the present invention relates to live attenuated vaccine to protect animal or human against infectionPasteurella multocidaor its pathogenic effects, which contains live attenuated bacterium according to the invention and a pharmaceutically acceptable carrier.
These vaccines include immunogene effective amount of a live attenuated bacterium according to the invention. “And monogene effective” means, the number of live attenuated bacteria introduced when vaccination is sufficient to induce the owner of an effective immune response against virulent forms of bacteria.
In addition immunogene effective amount of a live attenuated bacterium described above, the vaccine according to the invention also includes pharmaceutically acceptable carrier. The specified media may be as simple as water, but it can also include, for example, the culture fluid in which the cultured bacteria. Other suitable media include, for example, a salt solution at physiological concentrations.
A suitable dose for administration will vary depending on the age, body weight and vaccinated animal, the route of administration and the type of pathogen against which the vaccine. The examples below give an example of suitable dosages. The expert is able to extrapolate given the number of other species of animals.
The vaccine can contain any dose of bacteria sufficient to induce an effective immune response. Doses of less than 102live attenuated bacteria may not always be successful for adequate stimulation of the immune system, and doses of more than 1010live attenuated bacteria are not attractive to commercial the point of view.
Doses ranging from 103up to 109bacteria are typically quite appropriate doses.
Optionally, the vaccine can add one or more compounds with adjuvant activity. Live attenuated bacteriumPasteurella multocidaaccording to the invention does not necessarily require the specified Freund for efficiency, but in particular in the case of combined vaccines containing live attenuated bacteriumPasteurella multocidaaccording to the invention and antigenic material from another pathogenic virus or microorganism (see below) can be an advantageous addition of adjuvant.
Adjuvants are non-specific stimulators of the immune system. They enhance the immune response of the host body to the vaccine. Well-known specialists adjuvants are complete and incomplete beta-blockers, vitamin E, non-ionic block polymers, muramyldipeptide, ISCOM (immunostimulating kits, relatively, for example, European patent EP 1099 42), saponin, mineral oil, vegetable oil, and carbopol.
Adjuvants, particularly suitable for application to the mucous membranes are, for example, thermo-labile toxin (LT)E. colior cholera toxin (CT).
Other suitable adjuvants are, for example, aluminum hydroxide, aluminum phosphate or aluminum oxide, oil the e emulsion (for example, Bayol F® or Marcol 52®), saponins or solubilized vitamin E. the Use of these adjuvants is especially preferred when added to other virus or subunit, vaccines, for example, in the case of combination vaccinesPasteurella multocida.
Thus, in the preferred form of this variant implementation of the present invention live attenuated vaccine according to the invention contains an adjuvant.
Other examples of pharmaceutically acceptable carriers or diluents suitable for the present invention include stabilizers, such as SPGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein containing protein agents, such as serum of cattle or skim milk, and buffers (e.g. phosphate buffer).
Especially when these stabilizers are added to the vaccine, the vaccine is very suitable for lyophilization. Lyophilization has the advantage that the dried material does not require special conditions of storage, such as storage at temperatures below -20 or even -80 degrees Celsius. Thus, in the preferred form of implementation of the present invention live attenuated vaccine is presented in lyophilized form.
The vaccine containing the bacteria is a theory according to the invention, as described above, it is advantageous to include antigens derived from another microorganism or virus that is pathogenic to humans or animals, or antibody against the specified antigen.
There are several ways to manufacture a specified vaccine. One easy approach is to mix a live attenuated strain ofPasteurella multocidaaccording to the invention with one or more antigens from other pathogens of human or animal and a pharmaceutically acceptable carrier. So, even more preferred form of the present invention relates to live attenuated vaccine according to the invention, which differs in that it additionally contains one or more antigens selected from the group of viruses and microorganisms pathogenic for humans and/or animals.
Preferably, the antigens are selected from the group described above pathogens of pigs, cattle or poultry.
Another variant implementation of the present invention relates to live attenuated bacterium according to the invention for use in the vaccine.
Another variant implementation of the present invention relates to the use of live attenuated bacterium according to the invention for the production of vaccines to protect human or animal against infection by the bacteriumPasteurella ultocida or pathogenic effects specified infections.
For the introduction of an animal or human vaccine according to the invention it is possible to introduce, among other ways, intranasally, intradermally, subcutaneously, orally, by spraying or intramuscularly. For use in poultry oral administration (drinking water), sputtering and instillation in the eye are especially appropriate in only the simplicity of these routes of administration.
The specialist will know how to administer the vaccine according to the invention, since the method will not be significantly different from the methods of vaccination existing vaccinesPasteurella multocidaespecially attenuated vaccinesPasteurella multocida. The vaccine according to the invention, especially for poultry, should preferably be administered orally with drinking water, or by spraying.
Another variant implementation of the present invention relates to a method of making a vaccine according to the invention. These methods include mixing the live attenuated bacterium according to the invention and a pharmaceutically acceptable carrier.
Taking into account the fact that diseases caused byPasteurella multocidain most cases, are highly contagious, it would be highly advantageous to have a quick and simple tool, diagnostic industries, the ski test for early detection of infectionPasteurella multocidaanimals.
These diagnostic tests must be both fast and selective, in the sense that they should provide early identification of, and they must be specific toPasteurella multocidaand not to give false-positive reactions with other bacteria, regardless of whether other bacteria to other species ofPasteurellaor to the species, not related toPasteurella.
Diagnostic tests based on the presence or absence of antibodies againstPasteurella multocidaalthough frequent and reliable, are not very attractive, if you want early identification of bacteria. This is because the production of antibodies againstPasteurella multocidathe infected animal may take time up to two weeks.
Thus, another aim of the present invention is to develop diagnostic tools that are appropriate for early and specific detection of infectionPasteurella multocida.
Currently, it has been unexpectedly found that the sequence of the gene Orf-15 is unique toPasteurella multocidaand it is not present in other members of thePasteurellaceae.
Thus, another variant of implementation of the present invention relates to the tests based on RNA or DNA to identify thePasteurella multocida.
Diagnostic test for vyyavleniya> Pasteurella multocidabased on, for example, on the reaction of DNA or RNA extracted from the subject animal, with specific probes, or he represents, for example, (-) PCR-based gene sequence Orf-15 or on the basis of sequences of nucleic acids that are complementary sequence. If the molecules of nucleic acids that are specific for proteins associated withPasteurella multocidaaccording to the invention, are present in the animal, they will, for example, a specific way to communicate with specific PCR primers and subsequently will amplificates during the reaction (-) PCR. The PCR reaction product can then be easily identified during electrophoresis, the DNA in the gel. The reaction of (-) PCR is well known in the art (see link below). Molecules of nucleic acid most just can be isolated from the affected tissues or body fluids of the subject animal. In pigs smear from a nose provides suitable material from the affected lung tissue for research (-) PCR. The tracheal aspirates or material from diseased tissue liver, lung or heart are the preferred source of chickens. At Buffalo, sheep and cattle authorities of choice are the nose and diseased tissue lung.
Standard guidelines for PCR describes how to determine the length of the Prime is the moat for the selective PCR reactions with molecules of nucleic acids, specific gene Orf-15, according to the invention. Often used primers with a sequence of at least 12 nucleotides, but the primers longer than 15, preferably greater than 18 nucleotides are to some extent more selective. Especially widely used primers with a length of at least 20, preferably at least 30 nucleotides. Methods PCR is described (Dieffenbach & Dreksler, PCR primers, a laboratory manual. ISBN 0-87969-447-5 (1995)).
Molecules of nucleic acids gene Orf-15 or some of the molecules of nucleic acids having a length of at least 12, preferably 15, more preferably 18, even more preferably, 20, 22, 25, 30, 35 or 40 nucleotides, in ascending order of preference, or molecules of nucleic acids complementary to them, are thus part of the invention. These molecules are nucleic acids can, for example, be used as primers in the reaction (-) PCR to increase the number of nucleic acid that encodes a protein according to the invention. This allows you to quickly amplify a specific sequence of nucleotides for use as a diagnostic tool, for example, to identify thePasteurella multocidain tissue, as described above.
PCR reactions as hybridization reaction, well known in the art and, among PR is sneeze, described in Maniatis/Sambrook (Sambrook, J. et al. Molecular cloning: a laboratory manual. ISBN 0-87969-309-6).
Thus, another variant of implementation of the present invention relates to a diagnostic test for the detection of DNA or RNA associated withPasteurella multocida; specified test has the distinguishing feature that it comprises a nucleic acid molecule having the nucleic acid sequence depicted in SEQ ID NO:1, or a nucleic acid molecule that is complementary to a specified nucleic acid sequence or its fragment having a length of at least 12, preferably at least 15, more preferably at least 18 nucleotides.
Selection of mutant P-1059, spontaneously resistant to nalidixic acid
The strain ofPasteurella multocidacultivated in broth Luria-Bertani (LB) at 37°C over night. 0.2 ml of the culture was distributed on LB agar cups containing nalidixic acid 10 mg/ml, and incubated at 37°C for 48 hours. Took a couple of drug-resistant colonies and newly seeded on LB agar Cup containing nalidixic acid. After 48 hours incubation one resistant colony was selected and was inoculable in 10 ml of LB broth containing 20 mg/ml nalidixic acid, and cultured overnight. The culture was mixed with 5 ml of g is of acerina, selected aliquots of 1.8 ml tubes (1 ml per tube) and stored at -70°C. the Strain was resistant to nalidixic acid, identified as R-1059NR.
Breeding spontaneousthyAmutant R-1059NR
R-1059NR were cultured in LB broth containing 20 mg/ml nalidixic acid and 10 mg/ml thymine at 37°C over night. 0.2 ml of the culture was distributed on LB agar cups containing 20 mg/ml nalidixic acid, 10 mg/ml trimethoprim and 50 mg/ml thymine, and incubated at 37°C for 24-48 hours. Ten colonies were transferred to the same Cup LB Cup, which contained only 20 mg/ml nalidixic acid and 10 mg/ml of trimethoprim. Colonies that grew on plates of the first kind, but not the cups of the second kind, was athyA-mutants. One of thethyAmutants were inoculable in 10 ml of LB broth containing 20 mg/ml nalidixic acid and 150 mg/ml thymine, and incubated at 37°C over night. The culture was mixed with 5 ml of glycerol were selected aliquots of 1 ml per tube and stored at -70°C. was KeptthyAmutant identified as R.
Designing Tn vector pYL1.3
GenethyAE. coliamplified using polymerase chain reaction (PCR) from genomic DNA ofE. coliK-12 using primers 5'-AAGCTTGGCTGTCTCAGGTTTGTTCC-3' and 5'-TAGCTTGGCCAGTTTCTATTTCTTCG-3'. PCR fragment timbervale using T4 DNA poly is erase plus dCTP. pLOF/Ptt (Herreno, M., de Lorenzo, V., Timmis, K.N., J. Bacteriology, 172, 1990, 6557-6567) were digested using Xba I and Sf for deletion of the gene Ptt, and partially filled enzyme maple and dCTP. Trimmermania PCR fragment (one end) ligated with partially filled by the end of the Xba I cleaved plasmids. The other end of the PCR fragment and the end of the Sfi cleaved plasmid was a small mistake using T4 DNA polymerase and dNTP. This treated plasmid with legirovannym PCR fragment was samsarically and transformed into SM 10E. coli. One transformat containing plasmid suitable size, cleared, and cultivated, selected aliquots and stored at -70°C. Plasmid in the specified transformance meant as pYL1.3.
Designing libraries Tn mutantsP.multocida
R were cultured in LB broth + 200 mg/ml thymine at 37°C with vigorous shaking for approximately 48 hours.E. coliSM10 containing pYL1.3, were cultured in LB broth overnight. 0.1 ml R and culture SM10E. colimixed, were seeded on LB + (IPTG 100 mg/ml + 10 mm MgSO4+ thymine 200 mg/ml) and incubated at 37°C over night. The bacterial lawn was washed with cups and collected. 0.1 ml of the collected suspension was seeded on a Cup of LB + nalidixic acid 20 mg/ml and incubated at 37°C for 48 hours. Approximately 150 transconjugate collected and re-seeded on Cup LB + n is litikova acid 20 mg/ml for cleaning. These transconjugate were cultured in LB broth + nalidixic acid 20 mg/ml and stored at -70°C. These transconjugate re-seeded at the same time for a Cup of LB + nalidixic acid 20 mg/ml and cups LB + nalidixic acid 20 mg/ml + ampicillin 25 mg/ml for selection transposones mutants. Transconjugate (about 120), which grew only on the cups LB + nalidixic acid 20 mg/ml, rekultivirovana in LB broth + nalidixic acid 20 mg/ml, were selected aliquots and stored at -70°C.
Response (southern blotting) transposones mutants
The number 17 Tn mutants were selected in a random way and were cultured in LB broth + nalidixic acid 20 mg/ml Genomic DNA mutants were extracted using a kit QIAmp kit (QIAGEN Inc., Valencia, CA, USA). DNA was digested using Hind III. Genomic DNA P-1059 were digested using Hind III, pGP704 (Herreno, M., de Lorenzo, V., Timmis, K.N., J. Bacteriology, 172, 1990, 6557-6567), and also included pYL1.3 as controls. Southern blotting was performed by standard methods (Sambrook, J. et al., ed., Molecular cloning, 2nd edition, Cold Springs Harbor Laboratory Press, Plainview, NY, 1989). Tagging DNA DIG and directiony set (Poche Molecular Biochemicals, Indianapolis, IN, USA) was used to test labeling and southern blot. pGP704 and PCR amplificatory genethyAE. colimarked by digoxigenin and cleaved genomic DNA was probed with the according to the manufacturer's instructions. The results showed that most of the mutants was transpositionally mutants with a single insertion of Tn, and a small number of mutants was a mutant with the integrated plasmid.
Transposone mutants obtained in the described manner, checking on the subject attenuated behavior during standard experiments on animals.
Those transposones mutants, which were attenuated, the site of insertion of the transposon was identified and sequenced the broken gene.
One of the detected transposones mutants designated as strain RPasteurella multocida(briefly, strain R-15). This mutant had a transposon inserted into the Orf-15. this strain was used for experiments with vaccination in the following example.
Experiments with vaccination
In these experiments, vaccination was carried out in conjunction with vaccination against Newcastle disease. This was done because vaccination vaccinePasteurella multocidaand vaccination against Newcastle disease in veterinary practice it is preferable to carry out in the same day, even in one and the same moment. The advantage of the experimental approach described in this document, therefore, is that it additionally gives an idea about the behavior of the vaccine in the field.
Kul is URS vaccine RM
Fresh culture of strain RPasteurella multocidain TEV used for aerosol vaccination. The infectivity titer was determined immediately after use. Culture of strain RPasteurella multocidaused to premirovany consisted of 1.5×108CFU/ml, and culture, used for active immunization consisted of 1.6×109CFU/ml
For the introduction of drinking water, 500 ml of each culture was centrifuged and centrifuge the precipitate was dissolved in 500 ml of a solution of skim milk. The culture of strain RPasteurella multocidaused to premirovany consisted of 1.2×108CFU/ml, and culture, used for active immunization consisted of 1.3×109CFU/ml
Culture of ND vaccine contained 7,3 EID50(infectious dose for eggs) per dose for one bird.
The turkeys were given food and waterad libitum.
The division into groups and dosing
|The number of birds||Vaccination at 14 and 28 days||The number of birds infected with virulent P.m. on day 42|
|25||R spray + vaccine ND||15|
|25||R in drinking water + vaccine ND||16|
See table 1 depicts the scheme of vaccination. All birds were vaccinated with spray vaccine ND using aerosol spray can. The vaccine strain R was carried out by spray using a spray gun (birds remained in the aerosol within 10 minutes when off air circulation) or drinking water. For vaccination with drinking water to 500 ml of fresh culture was centrifuged and resuspendable in 500 ml of 2% skim milk (20 g skim milk per liter of water). Water from turkeys were removed at least 6 hours prior to vaccination. 500 ml of vaccine culture was placed in a column with drinking water. After vaccination was resumed normal supply of drinking water.
Control infectionPasteurella multocida
Controlling infection was performed using intramuscular injections (1.0 ml, in the chest) fresh diluted culture of the strain ofPasteurella multocidawild-type serotype 1 containing 1.5 x 105CFU/ml of Culture for the control of infection produced in TEV in the form of a fresh five-hour cultures.
Mortality was recorded daily for days. The dead birds, and other birds through 7 days after infection, an attempt was made to highlightP.multocidafrom the liver.
Mortality was compared using Fisher's exact test (Statistix for Windows, version 2.0).
The results of vaccination
|R drinking water||0/25|
Results after controlling infection
|Vaccine group||Mortality||Re-allocation of vaccine strain|
|R drinking water||3/16||3/16|
Bold:significant difference with controls
Results and discussion
Surveillance after vaccination are summarized in table 2. After vaccination, and to control infection 2 birds died in the group R aerosol 0 in the group R drinking water and 0 in the control group. This mortality may have been related to the vaccine strains, as in the control group mortality was not observed. An autopsy after vaccination showed that the aerosol path induced several more pathology (damage lungs of air bags, which could be caused by vaccine strains), compared with a group of drinking water.
Monitoring after control of infection are summarized in table 3 and in the drawing. After lethal heterologous control of infection (1000×LD50) were set different levels of protection. Aerosol path vaccination was the most effective way (100%), then follows the path of vaccination via drinking water (81%).
From the presented results we can conclude that the mutant Orf-15Pasteurella multocidaaccording to the invention are very suitable strains for use as a live attenuated strain in the vaccine. Mortality after vaccination was observed at the highest possible dose (>109CFU/ml).
Remember that ka is dose vaccination and dose control of infection used in this experiment is high. The dose of the vaccine can be greatly reduced to a level that will not mortality or signs of disease. Also you can greatly reduce the dose control of infection (1000×LD50).
From the presented results we can conclude that the mutant Orf-15Pasteurella multocidaaccording to the invention provide a very good basis for safe and effective live attenuated vaccinesPasteurella multocida.
Description of figures
The drawing shows a comparison of cumulative mortality among the vaccinated animals (vaccination via drinking water and aerosol vaccination) and among control animals after control of infection.
1. Live attenuated Pasteurella multocida used as vaccines, where this bacterium is unable to Express a functional protein Orf-15 due to mutations in the gene Orf-15, and the said mutation comprises an insertion and/or deletion.
2. Live attenuated bacterium according to claim 1, characterized in that the bacterium carries a heterologous gene that encodes one or more antigens selected from the group of viruses and microorganisms pathogenic for humans and/or animals.
3. Live attenuated bacterium according to claim 2, characterized in that the specified Goethe is aalogichny gene inserted into a gene, encoding the Orf-15.
4. Live attenuated bacterium according to claim 2, characterized in that the specified one or more antigens selected from the group consisting of a virus reproductive respiratory syndrome swine (PRRS)virus pseudoleskeella virus, swine influenza, swine parvovirus, a virus, infectious gastroenteritis, rotavirus, swine circovirus 1 or 2, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Haemophilus parasuis, Mycoplasma hyopneumoniae and Streptococcus suis.
5. Live attenuated bacterium according to claim 3, characterized in that the specified one or more antigens selected from the group consisting of a virus reproductive respiratory syndrome swine (PRRS)virus pseudoleskeella virus, swine influenza, swine parvovirus, a virus, infectious gastroenteritis, rotavirus, swine circovirus 1 or 2, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Haemophilus parasuis, Mycoplasma hyopneumoniae and Streptococcus suis.
6. Live attenuated bacterium according to claim 2, characterized in that the specified one or more antigens selected from the group consisting of herpes virus bovine virus viral diarrhea in cattle, parainfluenza virus type 3, paramyxovirus cattle, virus, Pasteurella haemolytica, Staphylococcus aureus, Staphylococcus uberis, Escherichia coli, respiratory syncytial virus, bovine Theileria parva, Theileria annulata, Babesia bovis, Babesia bigemina, Babeia major, Trypanosoma species, Anaplasma marginale, Anaplasma centrale or Neospora caninum.
7. Live attenuated bacterium according to claim 3, characterized in that the specified one or more antigens selected from the group consisting of herpes virus bovine virus viral diarrhea in cattle, parainfluenza virus type 3, paramyxovirus cattle, virus, Pasteurella haemolytica, Staphylococcus aureus, Staphylococcus uberis, Escherichia coli, respiratory syncytial virus, bovine Theileria parva, Theileria annulata, Babesia bovis, Babesia bigemina, Babesia major, Trypanosoma species, Anaplasma marginale, Anaplasma centrale or Neospora caninum.
8. Live attenuated bacterium according to claim 2, characterized in that the specified one or more antigens selected from the group consisting of avian pox virus, infectious bronchitis virus, infectious bursitis virus, Marek's disease, agent anemia chickens, avian reovirus, Mycoplasma gallisepticum, a virus of turkeys, Haemophilus paragallinarum, poxvirus chickens, avian encephalomyelitis virus, duck plague virus, Newcastle disease virus, syndrome dropping eggs, virus, infectious laryngotracheitis, herpes virus of turkeys, Eimeria species, Omithobacterium rhinotracheale, Mycoplasma synoviae, Clostridium perfringens, Salmonella species and E. coli.
9. Live attenuated bacterium according to claim 3, characterized in that the specified one or more antigens selected from the group consisting of avian pox, VI the USA infectious bronchitis, virus infectious bursitis virus, Marek's disease, agent anemia chickens, avian reovirus, Mycoplasma gallisepticum, a virus of turkeys, Haemophilus paragallinarum, poxvirus chickens, avian encephalomyelitis virus, duck plague virus, Newcastle disease virus, syndrome dropping eggs, virus, infectious laryngotracheitis, herpes virus of turkeys, Eimeria species, Ornithobacterium rhinotracheale, Mycoplasma synoviae, Clostridium perfringens, Salmonella species and E. coli.
10. Live attenuated bacterium according to claim 1 for use in the vaccine.
11. Live attenuated vaccine to protect animals or humans from Pasteurella multocida infection or pathogenic effects, characterized in that the vaccine comprises a live attenuated bacterium according to claims 1 to 9 and a pharmaceutically acceptable carrier.
12. Live attenuated vaccine according to claim 11, characterized in that it includes an adjuvant.
13. Live attenuated vaccine according to claim 11 or 12, characterized in that it is in lyophilized form.
14. Live attenuated vaccine according to claim 11, characterized in that it further comprises one or more antigens selected from the group of viruses and microorganisms pathogenic for humans and/or animals.
15. The use of live attenuated bacterium according to claims 1 to 9 for the manufacture of a vaccine to protect humans or animals from infection by the bacterium Pasteurella multocida and the pathogenic effects of infection.
16. A method of manufacturing a vaccine according to § § 11 to 14, characterized in that the method includes mixing the live attenuated bacterium according to claims 1 to 8 and a pharmaceutically acceptable carrier.
SUBSTANCE: genetic predisposition is determined by analysing polymorphism of genes ACTN3 (R577X), CNB (5I/5D), AMPD1 (C34T) through a multiplex polymerase chain reaction. For this purpose, a set of primers is used, wherein for the gene ACTN3 (R577X) the primes used are ACTGCTGCCCTTTCTGTTGCCT-3' and S'-CTGCAGGTGGCACTGACCATA3', for the gene CNB (5I/5D) the primers used are 5'-GGAGTTTAAAAGCCAGCCAGTCATACTA-3' and 5'-TGGAAGATCACACCATTTGATTAGCAGT-3', for the geneAMPD1 (C34T) the primers used are S'-GCAATCTACATGTGTCTACCCCAAAG-3' and 5'-CACTGCTGAAAAATAGCCATGTTTCTG-3'. Further, 3 pairs of primers with average melting point of 58-59°C are merged in a single test tube at standard conditions. The reaction product is analysed through analysis of polymorphism of the length of restriction fragments.
EFFECT: invention provides a simple, cheap, specific method which enables to identify functionally significant polymorphous gene loci, grow the number of analysed genes for further introduction into any standard clinical laboratory.
2 cl, 2 dwg, 3 tbl, 1 ex
SUBSTANCE: present invention can be used during DNA diagnostics in medicine, veterinary, sanitary and epidemiological analysis and in criminalistics to identify criminals. The method of amplifying specific nucleic acid fragments employs thermally stable DNA polymerase having chain-displacement activity. Also, the present method employs direct and reverse primers in form of head-to-tail arranged tandem recurrent sequences of the main primer. The present invention enables to amplify specific DNA or RNA fragments with increasing multiplication factor for each cycle.
EFFECT: shorter reaction time and high sensitivity of the reaction.
4 cl, 8 dwg, 2 ex
SUBSTANCE: to determine the type of a potato plant, DNA is extracted from fresh plant material and analysed through PCR analysis. PCR analysis is carried out using a set comprising a biological marker, a reaction mixture consisting of 160 mcM of each dinucleotide triphosphate dNTP, 1.6 mM MgCl2, 0.3 mcM of each primer from the set, 0.3 units of a Taq-polymerase enzyme, 1x buffer for the Taq-polymerase and standard DNA of known potato types in amount of 50 ng. Polymorphous DNA markers are obtained and viewed using gel electrophoresis. In order to carry out titration of alleles extracted in the examined group, allele spectra of the analysed and control groups of samples are compared. The biological marker, which contains polymorphous DNA, is a specific nucleotide sequence obtained using a diagnostic set of three pairs of primers.
EFFECT: marker characterises the type of widespread and essential potato types, and samples of related species Solarium, most frequency used in crossing when creating new potato types.
5 cl, 3 dwg, 4 tbl, 3 ex
SUBSTANCE: claimed is method of determining hypermethylated CpG is the region of suppressor genes of tumour growth in human DNA, which includes obtaining of samples of highly purified DNA; fragmentation of isolated DNA with restriction endonuclease which does not have a recognition site in amplified region, in particular TaqI; hydrolysis of fragmented DNA with methyl-dependent site-specific endonucleases GlaL and Blsl, carried out in two separate samples; further amplification of hydrolysis products with application of a pair of primers, flanking gene area which contains CpG islands and making a conclusion on the basis of changes in comparison with control, determined in at least one of the two test samples during their electrophoretic separation.
EFFECT: invention method ensures possibility of efficient diagnostics of cancer diseases.
2 cl, 3 dwg, 3 ex
SUBSTANCE: amplification of target products in carried out in special DNA thermocycler, provided with a special reaction thermal unit. Said thermocycler provides in reaction vessels, which are represented by standard polypropylene test tubes, temperature ingredients, oriented at angle to direction of gravitation action. Time of said amplification constitutes 1-5 minutes.
EFFECT: invention makes it possible to accelerate PCN by means of convection and simplify its carrying out preserving high specificity.
4 cl, 2 dwg, 2 ex
FIELD: veterinary science.
SUBSTANCE: method to produce animal fodder consists in identification of group of breeds, to which this animal belongs, on the basis of its genome, and to select fodder for an animal. Formula of specified fodder at least complies with food necessities of animals that belong to this group of breeds. Specified method may also include production of fodder by composition of ingredients containing biologically active components in quantities and ratios corresponding to formula of this fodder.
EFFECT: using this invention will make it possible to develop and prepare animal fodders on the basis of these animals' genomes.
14 cl, 1 dwg, 19 tbl
SUBSTANCE: plants and seeds of soya, including genetic transformation MON89788, are resistant to glyphosate. Methods are also disclosed to determine presence of DNA molecules in sample, which code specified genetic transformation.
EFFECT: high resistance to glyphosate.
24 cl, 3 dwg, 3 tbl, 3 ex
SUBSTANCE: first plant or its part is exposed to infection dose of ToTV. Then plants with no or slight symptoms of disease are identified.
EFFECT: plants identified in such a manner as resistant to virus are used as donor ones to cross with recipient plants, and plants resistant to ToTV are chosen from descendant plants.
8 cl, 7 dwg, 5 tbl, 2 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biochemistry, particularly to a set of primers for amplifying the L1 gene of the human papilloma virus (HPV), a set for detecting the human papilloma virus (HPV) genotype and methods of detecting human papilloma virus (HPV) genotypes. The method involves primary PCR amplification of the HPV L1 gene in an analysed sample using a set of direct and reverse primers which are specific for the L1 gene section. Further, secondary PCR amplification of the product of primary PCR amplification of the L1 gene is carried out using a reverse primer to obtain a biotin-labelled single-stranded L1 gene. The product of secondary PCR amplification and biotin-labelled single-stranded L1 gene undergo a hybridisation reaction with one or more probes for detecting the HPV genotype or a probe for detecting the HPV genotype. Further, the product of the hybridisation reaction reacts with phycoerythrin, bonded with streptavidin. The level of fluorescent substance is measured and probes are identified in the product of the hybridisation reaction to identify the HPV genotype. Further, the HPV genotype is determined, as well as the level of its presence in accordance with probes and the level of the fluorescent substance.
EFFECT: invention provides a method for detecting HPV in a sample with high sensitivity, sufficient for detecting extremely small quantities of HPV in the sample.
8 cl, 2 dwg, 1 tbl, 4 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to microbiology. Described is a method for detecting living cells of microorganisms in a test sample, involving adding a cross-linking agent to the test sample, capable of cross-linking DNA when exposed to light having wavelength 350-700 nm; exposure to light having wavelength 350-700 nm; removing the cross-linking agent; adding medium and further incubation; adding cross-linking agent capable of cross-linking DNA when exposed to light having wavelength 350-700 nm to the incubated test sample once more; repeated exposure to light having wavelength 350-700 nm; extraction of DNA from this test sample and amplification of the target region of the extracted DNA and analysis of the amplified product. Disclosed is a method of detecting living cells of microorganisms in a test sample, which involves using a set containing the following components: cross-linking agent capable of cross-linking DNA when exposed to light having wavelength 350-700 nm; medium and primer(s) for amplifying the target region of the DNA of the microorganism which is the detection target using a nucleic acid amplification method.
EFFECT: invention enables detection of living cells of microorganisms in a test sample.
26 cl, 5 dwg, 17 tbl, 5 ex
SUBSTANCE: polypeptides exhibit antimicrobial activity and recovered polynucleotides coding these polypeptides. Nucleic acid constructs, vectors and host cells contain said polynucleotides.
EFFECT: applicability of polypeptides in veterinary science and medicine, as well as in forage production.
16 cl, 6 tbl, 7 ex
SUBSTANCE: plasmid vector pE-Trx-Aur is constructed for expression of aurelin in cells of Escherichia coli in composition of hybrid protein Trx-Aur, consisting of two DNA fragments, whose nucleotide sequence is given in description. By means of said vector parent strain of Escherichia coli is transformed, obtaining strain-producent of hybrid protein Trx-Aur. In order to obtain peptide aurilin cultivation of cells of obtained strain-producent is carried out, after that, performed are: cell lysis, affine purification of hybrid protein Trx-Aur on metal-chelate carrier, decomposition of hybrid protein Trx-Aur with bromine cyan by residue of methionine, introduced between sequences of aurelin and thioredoxin, and purification of target peptide by method of reversed-phase HPLC.
EFFECT: invention makes it possible to obtain biologically active aurelin by simplified technology and without application of hard-to-obtain natural raw material.
3 cl, 4 dwg, 1 tbl, 4 ex
SUBSTANCE: method of prevention and method of treatment of gastrointestinal diseases in animals or people, such as diarrhea, antibiotics-associated diarrhea, inflammatory intestinal disease and diarrhea, caused by Clostridium difficile (CDAD), includes stage of introduction of efficient amount of strain of Bacillus subtilus ATCC PTA-6737, as probiotic. Strain produces lipopeptide sufractine and represents medication for treatment of gastrointestinal diseases in animals or people, such as diarrhea, antibiotics-associated diarrhea, inflammatory intestinal disease and diarrhea, caused by Clostridium difficile (CDAD).
EFFECT: invention ensures high efficiency in treatment and prevention of gastrointestinal diseases in people or animals.
15 cl, 14 dwg, 12 tbl, 4 ex
SUBSTANCE: invention represents method to produce useful metabolite by fermentation of mix of primary source of carbon and secondary source of carbon, using bacterium of Enterobacteriaceae family, modified so that flows for recycling of primary source of carbon, such as carbohydrates or glycerin, and secondary source of carbon, such as ethanol, are separated in this bacterium. Method includes cultivation of bacterium in nutrient medium and extraction of metabolite from culture broth, besides, bacterium has increased expression of mutant alcohol dehydrogenase resistant to aerobic inactivation, reinforced with expression of mutant gene rthA, giving resistance to high concentrations of threonine, by deletion of gene coding threonine dehydrogenase, and is capable of production of useful metabolite selected from group consisting of glutamate, proline, threonine, lysin, aspartate, asparagine, methionine, serine, valine, leucine, isoleucine, cysteine, arginine and pyrimidines, by fermentation of mixture of primary source of carbon, such as carbohydrates or glycerin, and secondary source of carbon, such as ethanol, and is additionally modified so that flows of carbohydrates or glycerin and ethanol are divided through weakening of expression of gene (genes), coding their pyruvate kinase.
EFFECT: invention makes it possible to produce useful metabolites with high extent of efficiency.
13 cl, 2 dwg, 1 tbl, 9 ex
SUBSTANCE: method includes growing bacterium of Enterobacteriaceae family, which is modified by introduction of yeast gene ARO1 and extraction of aminoacid. Invention also relates to method for production of ester of lower alkyls of α-L-aspartyl-L-phenyl alanine, including growing specified bacterium, accumulation of L-phenyl alanine and synthesis of ester of lower alkyls of α-L-aspartyl-L-phenyl alanine from asparaginic acid or its derivatives and produced L-phenyl alanine.
EFFECT: invention makes it possible to produce aromatic amino acids and esters with high extent of efficiency.
12 cl, 2 dwg, 2 tbl, 2 ex
SUBSTANCE: method for making Escherichia coli bacterium for producing 1,2-propanediol of an initial modified bacterium with the tpiA, gloA, aldA and aldB gene deletion, involves the cultivation of said initial modified bacterium in an appropriate culture medium containing a carbon source with applying the increasing potencies in such a manner that the culture medium preserves only the microorganisms with the growth rate being equal or exceeding the applied potency, for evolving said initial modified bacterium of one or more genes involved in a biosynthetic pathway of DHAP to methylglyoxal, then to 1,2-propanediol to produce evolving genes coding enzymes exhibiting higher activity of '1,2-propanediolsynthase', selection and recovery of one or more Escherichia coli bacteria exhibiting higher activity of '1,2-propanediolsynthase'. Also, the invention concerns initial microorganisms and thereby produced evolving microorganisms and the method for making 1,2-propanediol and acetone by cultivation of evolving microorganisms.
EFFECT: more efficient 1,2-propanediol production.
19 cl, 3 dwg, 3 tbl, 7 ex
SUBSTANCE: invention relates to a method for synthesis of purine nucleoside 5'-aminoimidazole-4-carboxamide riboside (AICAR) and a Bacillus subtilis strain VKPM V-10167 - producer of AICAR. AICAR is obtained by culturing modified Bacillus subtilis bacteria in a suitable culturing medium. The modified bacteria used are Bacillus subtilis bacteria, obtained through successive introduction of mutations purR, purH::EmR, PurΔL and PrpsF-prs.
EFFECT: disclosed invention widens the range of methods for microbiological synthesis of AICAR and enables to engineering of the strain which produces AICAR for realising this method.
2 cl, 1 dwg, 2 tbl, 11 ex
SUBSTANCE: method involves cultivation of a microorganism of Enterobacteriaceae family capable to produce L-amino acid, in a nutrient medium, and recovery of said L-amino acid from the culture liquid. Said microorganism is modified by the introduction of a DNA fragment including a pho regulon promoter and a structural gene coding L-amino acid biosynthesis enzyme ligated behind the promoter in such a manner that gene expression is controlled by said promoter. And the activity of L-amino acid biosynthesis enzyme increases in the promoter gene expression with phosphorus concentration in the medium is such that induction of said promoter is observed.
EFFECT: invention allows producing L-amino acids with high efficiency.
12 cl, 4 dwg, 7 tbl, 2 ex
SUBSTANCE: invention represents plasmid DNA pD4spGBD enabling the expression of a recombinant antigen - protein LigA L. interrogans domain 4 in chimeric protein D4-GBD and 1,3-β-glucan-binding domain (GBD), and a method for making a based subunit engineered leptospirosis vaccine.
EFFECT: invention allows ensuring the development of an intensive immune response and high level of antibodies synthesis with no additional adjuvants.
6 cl, 1 dwg, 6 ex
SUBSTANCE: invention represents plasmid DNA pD5spGBD enabling the expression of a recombinant antigen - protein LigA L. interrogans domain 5 in chimeric protein D5-GBD and 1,3-β-glucan-binding domain (GBD), and a method for making a based subunit engineered leptospirosis vaccine.
EFFECT: invention allows ensuring the development of an intensive immune response and high level of antibodies synthesis with no additional adjuvants.
6 cl, 1 dwg, 6 ex
SUBSTANCE: invention refers to veterinary microbiology. A vaccine contains inactivated bacterial suspension Pasteurella multocida of serological versions A, B, D and an adjuvant. Additionally, the vaccine contains inactivated bacterial suspension Pasteurella haemolytica with activity of soluble surface antigens in passive hemagglutination test 1:32-64, and as an adjuvant - the adjuvant ISA-70VG in the following proportions, wt %: inactivated bacterial suspension Pasteurella multocida of serological versions A, B, D and inactivated bacterial suspension Pasteurella haemolytica taken in the mass ratios 1:(0.8-1.2):(0.8-1.2):(0.8-1.2) - 45-55, the adjuvant ISA-70VG - the rest.
EFFECT: vaccine shows high immunologic activity and prolonged storage period.