The recombinant strain of vaccinia virus, excessively structural proteins of the virus venezuelan encephalomyelitis of horses and suitable for the production of immunobiological preparations and method thereof

 

(57) Abstract:

Usage: biotechnology, development of immunobiological preparations for the diagnosis and prevention of Venezuelan encephalomyelitis of horses. The essence of: obtaining a recombinant strain of vaccinia virus expressing the proteins of the virus Venezuelan encephalomyelitis of horses (VVEL). The strain is obtained by embedding in gene timedancing commercial strain of vaccinia virus (LIWP) under control of the promoter of the protein of 7.5 To vaccinia virus sequences encoding structural proteins of WEL (full-size DNA copy of the entire 26 S RNA). The result is a strain of vaccinia virus, which generated the most complete immune response against WAL. The infection of cells obtained by the strain on their surface expressed glycoproteins supercasino shell of the virus Venezuelan encephalomyelitis of horses. 2 S. p. f-crystals, 2 ill., table 2.

The invention relates to biotechnology, in particular genetic engineering, is a recombinant strain of vaccinia virus, causing the synthesis of structural proteins of the virus Venezuelan encephalomyelitis of horses (VAL) intelroom design of this strain.

Virus VAL is one of the most pathogenic for animals of the genus of alpha viruses collection Togaviruses that causes the most serious disease in humans, rodents and horses and leading for a wide range of animal species, to death. This virus is transferred in nature by several species of mosquitoes and causes large-scale epidemic in South, Central and North America.

For the prevention of disease caused by the virus VAL, currently applied vaccination of people and farm animals live vaccine based on attenuated strains TC-83 and 230, as well as inactivated vaccine, manufactured on the basis of the processed formalin virus VAL, however, the possibility exists that can reverse these strains to virulent variant [1] and the inclusion of revertants in natural circulation [2] in Addition, the strains TC-83 and 230 reactogenna (in 80% of cases) may have a teratogenic potential of the virus VAL [3] and 30% of vaccinated cause symptoms, similar symptoms VAL [4] Immunization same inaktivirovannye virus involves the use of large quantities of material, which leads to large-scale developments of the pathogen and the high is managing to Express foreign genes in eukaryotic cells. Created to date, vectors based on retroviruses, adenoviruses, papillomaviruses and papovaviruses have a number of disadvantages: low speed reproduction in cell culture, small capacity relative to the embedded genetic sequences, inability to multiply in the body of animals and others [5] Some progress on the development of recombinant vaccines have been achieved by using a vaccinia virus. In the genome of this virus has been integrated sequence of the hemagglutinin gene of influenza virus [6] N - and G-protein of vesicular stomatitis virus [7] the glycoprotein of rabies virus [8] antigen Plasmodium malaria [9] and others In all cases was observed effective expression alien to ospowiki proteins in cell culture. In some cases, in the body of the vaccinated animals were detected by antibodies specific to these proteins and can protect against lethal infection caused by a virus whose genes were integrated into the genome of the recombinant vaccinia virus. The potential expression of the structural proteins of alpha viruses in the genome of the recombinant vaccinia virus was demonstrated on the example of virus Sindbis [10] for expressing the structural proteins of the American variant strain Trinidad donkey virus VAL and derived vaccine strain TC-83 [11] which consists in the following:

1. Constructed a plasmid containing the gene sequence of the structural proteins under the control of the 7.5 K promoter of vaccinia virus, flanked on both sides by sequences of the gene timedancing (TC) of vaccinia virus.

2. Is recombination between "TC-shoulders derived plasmids and DNA genome into cells infected with vaccinia virus.

3. The selection of clones of recombinant vaccinia virus with phenotype TC-on the selective medium containing bromosuccinimide.

4. The selection methods of hybridization of clones ospowiki, with the phenotype TC-containing the genome sequence embedded gene.

5. The resulting clones are analyzed by cell culture to determine the expression level of the built-in sequences.

However, in each case, depending on the sequence introduced into the genome ospowiki, and a promoter which is selected to implement the expression that describes the steps of this method are so very different that it cannot serve as a universal. The most similar in structure from all the built-in sequence is a gene region studnie sequence 26 S RNA used in the prototype American and used by us domestic version pathogenic strain of the virus VAL Trinidad dunks have some differences, leading to amino acid substitutions in the encoded 26 S RNA proteins. In the protein C 62nd Ser Pro; in the protein 6K 48th Met to Val, 53 s Ala to Gly, 54th Pro Ala, 55-th Ala to Gly and additional Ala at position 56; protein E2 (main immunogen) 85th His to Tyr, 147-Oh Val to Ala, 187-th Thr on Jle, 192-Oh Val to Ala and 408-Oh Jle at the Met. To introduce genes for structural proteins VAL into the genome of vaccinia virus in the prototype are encouraged to use a restriction enzyme Tth III I, which results in the deletion of the embedded DNA copies of the 26 S RNA 3'-untranslated region with a poly-A tract. In addition, when using this restrictase at the 5'-end embedded DNA copy remains part of the sequence that encodes a non-structural protein nsp 4, which may hinder the efficient transcription of the sequence that encodes the structural proteins of the virus VAL. In the literature there is no analysis of the protective properties of recombinant virus VACC/TRD, carrying the insert 26 S RNA American variant strain Trinidad donkey virus VAL.

The aim of the invention is the creation of a recombinant strain of vaccinia virus that causes the synthesis of all the structural proteins of virulent strain TRD virus VAL in infected cells and providing effective protection vaccinated lab icescape strain of vaccinia virus-LIIT under the control of the promoter of the protein virus 7.5 K S sequence encoding structural proteins of the virus VAL (full-size DNA copy of the entire 26 S RNA), to retrieve from which DNA copies of genomic RNA domestic virulent variant strain TRD virus VAL) used restriction enzyme Apal, website recognition, which is located directly in front of the 5'-end subgenomic 26 S RNA and is unique to this sequence.

The resulting recombinant strain ospowiki contains the genome, compared to the original commercial opvattingen strain (LIIT), optional sequence, comprising: 1) from the fragment length of 300 N. p. including the promoter region of the gene protein 7.5 K; 2) from the fragment length of about 4000 p. O. containing the sequence of the protein coding genes C, E3, E2, 6K and E1 virus VAL, domestic variant strain Trinidad donkey, in the specified order; integrated DNA copy of the 26 S RNA has a 5'-end of the other sequences of the genomic RNA VAL, able to prevent transcription [12] Scheme built-in fragment shown in Fig. 1. It should be noted that insert a full-size DNA copy of the 26 S RNA virulent strain of the virus VAL leads to the formation of the most complete immune response against the virus VAL.

This stateliest genes nonstructural proteins, which may prevent later correct translation, then the above fragments containing the sequence of the promoter and structural genes of proteins VAL, collected as part of the single plasmids within a gene timedancing of vaccinia virus. To do this, use the following plasmids: 1) a plasmid-based vector pUC8 (pVE-4, pVET7-91, pVEI47), containing the sequence 5'-end of the 26 S RNA virus VAL length of at least 4000 p. O. and COOH-terminal region of the gene protein Nsp4; 2) a plasmid containing the cloned between the restriction sites SalGI and BamHI of polylinker SalGI-HinfI fragment of DNA of vaccinia virus strain WR, size 253 p. N. corresponding to the promoter of the gene of the protein of 7.5 To this virus [13] (denoted as p7,5K); 3) plasmid pTK1285 [14] contains the sequence of a gene timedancing of vaccinia virus, strain LIVP, with built-in polylinker.

This stage of designing a new strain is removed from any of the plasmids mentioned above in item 1 (e.g pVE4, pVET7-91 or pVE147) cDNA fragment corresponding to a region of non-structural proteins of the virus VAL. For this purpose, the plasmid is cut by a unique ApaI restriction site, directly adjacent to the beginning subgenomic 26 S RNA virus VAL, and one of the sa shall provide a DNA polymerase of phage T4 according to [15], followed by cyclization of the plasmid using DNA ligase of phage T4. After transformation of E. coli cells obtained ligase mixture selected clones, plasmids are not SalGI-ApaI fragment corresponding to a region of non-structural proteins of the virus VAL, the size of which can vary depending on the source selected from clonetech plasmids, and restore the site to know restrictase SalGI. Selected clones of E. coli produce a plasmid designated as pVE5.

Then DNA plasmids RTC hydrolyzing endonucleases, restriction SalGI and EcoRI, after which the hydrolysis product with the help of DNA ligase of phage T4 connect with fragments SalGI-BamHI plasmid P7,5K and BamHI-EcoRI plasmid pVE5. After transformation of E. coli cells grown from clones produce plasmid, designated as pVE5.1. This plasmid contains a gene timedancing of vaccinia virus, strain LIVP, in the coding part of which is built sequence of cDNA copies of the 26 S RNA virus VAL (with possible concomitant another fragment of the plasmid at its 3'-end) under the control of the promoter of the gene of the protein of 7.5 To vaccinia virus.

The second phase of the work consists in the implementation of recombination between the DNA of a commercial strain LIVP of vaccinia virus and TC-shoulders plasmids pVE5.1, which is achieved by cotransfection them monolayer cells CV-1, INFCIRC-the phenotype of the cell culture Human 143 (TC-in the presence of 5-bromo-2'-dose irradiation on neurogenesis and ability hybridisierung with a radioactive probe, complementary to the built-alien genes, prigotavlivaemy based plasmids pVE4. Further characterization of clones ospowiki is carried out by radiation products of its genes expression in the culture of infected cells with sera specific for surface proteins VAL, as well as by studying the protective effect against infection by the virus VAL arising by immunization of animals with recombinant vaccinia virus.

The resulting recombinant strain of vaccinia virus belongs to the family from the poxviridae genus classified in the genus orthopoxvirus and has properties typical representative of the genus orthopoxviruses. Has the cryptogram: T/2:160/5: X/*:Y/0.

Virions have the characteristic shape of the briquette size 200x300 nm and according to electron microscopy does not differ from the original strain of vaccinia virus LIVP, i.e. have nucleoproteins the core of a biconcave shape, the recesses which are so-called lateral calf. There are two types of Larionov: intracellular covered one lipoprotein envelope, and vneck Physico-biochemical characteristics and cultural properties of the strain.

The main components of the virion are: proteins ( 90%), lipids ( 5%) and DNA ( 4% ). Gene recombinant strain of vaccinia virus presents double-stranded DNA size 190000 p. N. which in the coding part of the gene timedancing contains DNA copy subgenomic 26 S RNA domestic variant of the virus VAL Trinidad dunks under the control of the promoter of the protein of 7.5 To vaccinia virus. In Hind III digests of the isolated viral DNA recombinant strain instead of Hind III-K-DNA fragment of the original strain LIVP there are two fragments having a size of about 800 and 9000 p. N. the latter of which according to DNA-DNA blot hybridization contains the genes encoded in subgenomic 26 S RNA virus VAL.

When the reproduction of the recombinant strain in chicken embryos, the nature of the lesions on chorioallantoic shell similar to the lesions formed by the strain LIVP, and after 48 h incubation at 37oC gives the drain defeat at the dose of 105O. O. E. embryo. In addition, the obtained recombinant strain is not significantly different from strain LIVP on productivity in the monolayer transplantable cell lines CV-1, Human 143 (TC-), Rat 2 and suspension culture VNC, giving the final credits, similar to the original title of vaccinia virus. what to find on transplantable cell lines Human 143 (TK-) and Rat 2 in the presence of 25 μg/ml 5-bromo-2'-dose irradiation on neurogenesis.

Pathogenicity for animals.

The study of the properties of the obtained recombinant strain of vaccinia virus intraperitoneal, subcutaneous and intraplantar the introduction of different doses of white outbred mice, as well as when intradermal rabbits showed that toxicity and necrotic activity of recombinant strain does not differ from the original strain LIVP.

The main significant difference recombinant strain from strain LIVP is the ability to Express on the plasma membrane of infected obtained strain cells glycoproteins supercasino shell of the virus VAL.

The recombinant strain of vaccinia virus VR26S with integrated DNA copy of the 26 S RNA domestic variant of the virus VAL Trinidad dunks are not described in literature and has significant differences from known strains of recombinant viruses. This strain is deposited in the State collection of viruses. The strain number is assigned by the depositor GKV N 944.

Significant differences of the proposed method of obtaining a recombinant vaccinia virus are as follows: for installation virionyx GE proteins the plasmid, containing the insert DNA, complementary to the COOH-terminal region of the gene of the protein NS4, beginning subgenomic 26 S RNA VAL and sequence that encodes the structural proteins, including genes of the protein, E3 and E2; adding to this sequence of protein coding genes 6K and E1 leads to more robust immune response to VAL in laboratory animals after vaccination, the virus recombinant ospowiki. The second significant difference between the proposed method is the use in the design process of restrictase ApaI. Website recognition of this restrictase is the gene sequence of the protein NS4 immediately before the beginning of the 26 S RNA and its use allows to split hampering the effective translation of the gene sequence of non-structural protein without using time-consuming procedures, such as, for example, hydrolysis by nuclease Bal 31.

The essential features of the obtained recombinant strain ospowiki are as follows:

first, the strain obtained on the basis of domestic commercial vaccine strain of vaccinia virus LIVP;

secondly, on the plasma membrane of cells infected with recombinant virus expressed glycoproteins supercasino which leads to the appearance in their blood high antibody titer, specific to the virus VAL, and protection from lethal infection caused by the subsequent injection of large doses of the virus VAL.

The essential feature of the method of obtaining a recombinant strain of vaccinia virus is that in the genome of the domestic commercial strain LIVP of vaccinia virus is embedded foreign genetic material, consisting of genes for structural proteins of the virus VAL, strain Trinidad donkey (Owls), under the control of the promoter of the gene of the protein of 7.5 TO RC. Selection of recombinant clones of strain is to have the effect of protecting immunized lab animals from lethal infection caused by large doses of pathogenic virus VAL.

In Fig. 1 shows the schematic design of the genome of strain recombinant vaccinia virus containing the DNA copy of the 26 S RNA virus VAL in the composition of the gene timedancing under the control of the promoter of the gene of the protein of 7.5 K.

Letters marked the sites of recognition the following restricts: E, EcoRI, B - BamHI, S SalGI, P > PST, A, ApaI, C, ClaI, X XmaI, H Hind III

The last line shows the schematic map of the restriction by the restriction enzyme Hind III genome of vaccinia virus.

In Fig. 2 radioimmunological analysis of samples using: (A) rabbit anticipate Human cells 143;

2.3 Human cells 143, taken after 4 and 8 h after infection, the parent strain LIVP of vaccinia virus;

4.5 Human cells 143, taken after 4 h after infection with recombinant clones 159 and 3110, respectively;

6,7 the same thing, but in 8 h after infection;

9 same as 3, but with the use of monoclonal antibodies;

10,11 same as 6, 7, but using monoclonal antibodies.

A method of obtaining a strain of recombinant vaccinia virus expressing the genes for structural proteins VAL, his achievements, storage and research immunogenic properties is illustrated by the following examples:

Example 1.

Cells of Escherichia coli containing plasmid DNA pVE4 (see Fig. 1), increasing in 100 ml of broth before saturation. Plasmid DNA secrete according to the standard technique. Conduct joint hydrolysis of 1 μg of plasmid DNA restrictase ApaI (10% act.) and SalGI for 3 h at 37oC; after incubation should the phenol deproteinization with subsequent alcohol precipitation.

Next are the completion of all of plasmid DNA to the dumb, carried out in buffer (0,033 M Tris-acetate pH 7,9; 0,66 M potassium acetate; 0.01 M magnesium acetate; 0.001 M 2-mercaptoethanol) DNA polymerase, phage T4 (1 unit and is of Shatov, then they add up to a concentration of 0.1 mm. After phenol deproteinization and alcohol precipitation of the plasmid cyclist using DNA ligase of phage T4 (0,1% act.) in buffer C (0.05 M Tris-HCl pH 7.5; 0.025 M MgCl2; 0.01 M NaCl; 0.005 M 2-mercaptoethanol; 0.1 mm ATP) for 1 h at 12oC.

The transformation of the cells is carried out as follows: 0.1 ml of cell suspension of E. coli jM 103 contribute in 20 ml of nutrient broth LB and grown to a titer 5108cells/ml Cells from 3 ml of medium harvested by centrifugation (3000 rpm, 10 min, 0oC), resuspended in 1 ml of buffer I (0.01 M MOPS pH 7.0; 0.01 M RbCl), precipitated by centrifugation, suspended in 1 ml of buffer B (0.1 M MOPS pH 6.5; 0.05 M CaCl2; 0.01 M RbCl) and leave for 15 min in ice. After that, cells are again collected by centrifugation, suspended in 200 μl of buffer I add 3 ál of DMSO, plasmid DNA in a volume of 10 μl and left in ice for 30 minutes After completion of the incubation, the cell suspension is heated for 30 s at 44oC, diluted 25-fold with LB broth, incubated 30 min at 37oC and plated on the agar LB medium containing ampicillin at a concentration of 25 ál/ml Of cells grown in 18 h clones, isolated DNA plasmids, designated by us as pVE5, by the method described above. This pay.

Hydrolysis of DNA plasmids: pVE5 (10 µg) restrictase BamHI and EcoRI, P7,5K (10 µg) restrictase SalGI and BamHI, RTC (10 µg) restrictase SalGI and EcoRI, carried out using 20 units of the act. each restricts, for 8 h at 37oC. After phenol deproteinization fragments of plasmids pVE5 and p7,5K were isolated by separation of the hydrolyzed DNA in 4% polyacrylamide gel followed by Electrosila on paper DE-81.

The connection of fragments of plasmids pVE5 and p7,5K in the structure of the plasmid RTC carried out in 10 μl of buffer With the above conditions. Vector linearized plasmid and restriction fragments are selected in such a way that the only option possible their mutual joining.

Received ligase mixture is used for transformation of E. coli cells, strain jM 108. Transformation conditions described above. From cells grown in 18 h clones, isolated plasmid DNA in analytical quantities and analyze inserts using restricted EcoRI, PST, SalGI and BamHI. About 80% of the clones contain the plasmid with the desired sequences. Of these clones produce plasmid, designated by us as pVE5.1, which after alkaline cleaning method and ravnovesnym ultracentrifugation is used for recombination with g the existing structural proteins of the virus VAL, monolayer culture of cells CV-1 infect the vaccinia virus strain LIVP (0,04-0,05 O. O. E./cell) and incubated in MEM medium containing 2% fetal calf serum (TC). After 2 h after the onset of infection on the monolayer of cells (25 cm2) put in 0.8 ml of calcium phosphate precipitate DNA pVE5.1.

The monolayer coated with coprecipitation incubated for 30 min at 37oC, pour to 7.2 ml of medium ΜM, containing 8% CU, incubated at 37oC for another 4 h, after which change the environment in 5 ml of fresh and continue incubation at 37oC for 48 hours and Then the cells twice freeze thaw, resuspended in the environment and thus obtained viral progeny are used for selection of recombinant virus variants S.

The selection is performed in several stages according to the method described in [5]

The first stage of selection is the cloning of viral progeny on the monolayer of cells Human 143 (TK-in the presence of 5-bromo-2'-dose irradiation on neurogenesis. When this is selected clones of the virus S, with the phenotype (TC-). The second stage of selection is to analyze (TK-) clones S method the dot-hybridization for the presence of viral genome inserted genes. [32P]-labeled probe prepared on the basis of the insertion PLA is about selected 2 clone recombinant virus S: v713-31 and v713-15 with integrated into the genome sequences of the genes for structural proteins VAL. After re-cloning of the virus from these clones and hybridization of DNA on a similar scheme with the same probe for further work were used clones v713-159 and v 713-3110. After for statement of additional control of recombinant virus by phenol extraction according to [5] was isolated genomic DNA, which is hydrolyzed in restrictases Hind III, followed by analysis of fragments of hydrolysis by electrophoresis in 0.8% of agarose gel according to [15] it was revealed additional fragment size of about 9000 p. O. containing the sequence of the gene timedancing, 26 S RNA and stI-EcoRI fragment of plasmid pBR322. Newly fragment hybridized with a radioactive probe, complementary genes for structural proteins of the virus.

The clones obtained recombinant virus S were accumulated on the cell culture KSS-21 and purified according to [11] These viral preparations were used to infect a monolayer of cells Human 143 to a plurality of 10-20 00E/cell. After just four hours after infection on the surface membrane of cells using solid-phase radioimmunoassay analysis described in [10] were identified structural proteins VAL. In these experiments, were used hyperimmune rabbit summer 2.

Cells of E. coli containing plasmid DNA pVET7-91 full-size DNA copy of the viral genome VAL, having at the 3'-end of the website to know restrictase Hind III, increase in 100 ml of broth before saturation. Plasmid DNA secrete according to the standard technique. Next, 10 μg of plasmid hydrolyzing the restriction enzyme ApaI (10% activity, 3 hours, 37oC) and after phenol deproteinization and alcohol deposition is completed the ends of the linearized plasmid DNA to the dumb, as in example 1. After phenol treatment and ethanol precipitation of plasmid DNA daresay the restriction enzyme Hind III and using gel electrophoresis and electroelution on paper DE-allocate 81 fragment 4000 p. O. according to the method of [15] Vector plasmid pUC8 linearized the restriction enzyme SalGI and complete its ends to a blunt fragment maple DNA polymerase from E. coli, according to [15] this was followed by further hydrolysis with restriction enzyme EcoRI. The resulting vector cyclist using DNA ligase of phage T4, as described in example 1 method, in the presence highlighted by restrictase ApaI fragment 4000 p. O. plasmids pVET7-91 and an additional EcoRI-Hind III polylinker fragment from plasmid pUC18 with size 55 p. O. Received ligase mixture of transformed E. coli cells grown is tons of them plasmid DNA, who analyze for the presence and correct orientation of installation using restricted BamHI and EcoRI.

Resulting from these genetic manipulations plasmid contains the sequence only DNA copies of the 26 S RNA virus VAL without excess 5'-terminal sequence and is fully equivalent plasmid pVE5 the location of the sites of recognition used further restricted (see diagram constructing recombinant viral genome in Fig. 1). DNA constructed plasmid is then used to obtain insertional plasmid pVE5.1, as described in example 1.

Example 3.

Cells of E. coli containing plasmid DNA pVE-147, carrier insertions 9800 3'-terminal bases of the DNA copy of the viral genome VAL and having at the 3'-end of the insertion site of recognition of restrictase Hind III, increase in 100 ml of broth before saturation. Plasmid DNA secrete according to the standard technique. Next, 10 μg of plasmid hydrolyzing the restriction enzyme ApaI (10% activity, 3 hours, 37oC) and after phenol deproteinization and alcohol deposition is completed the ends of the linearized plasmid DNA to the dumb as in example 1. After phenol treatment and ethanol precipitation of plasmid DNA daresay the restriction enzyme Hind III fornow plasmid pUC8 linearized the restriction enzyme SalGI and complete its ends to a blunt fragment maple DNA polymerase from E. coli, according to [15] this was followed by further hydrolysis with restriction enzyme EcoRI. The resulting vector cyclist using DNA ligase of phage T4, as described in example 1 method, in the presence highlighted by restrictase ApaI fragment 4000 p. O. plasmids pVE-147 and additional EcoRI-Hind III polylinker fragment from plasmid pUC18 with size 55 p. O. Received ligase mixture of transformed E. coli cells grown in the presence of ampicillin in LB-agar colonies develop in analytical quantities in LB-broth and separated from them plasmid DNA that analyze for the presence and correct orientation of installation using restricted BamHI and EcoRI.

Resulting from these genetic manipulations plasmid contains the sequence only DNA copies of the 26 S RNA virus VAL without excess 5'-terminal sequence and is fully equivalent plasmid pVE5 the location of the sites of recognition used further restricted (see diagram constructing recombinant viral genome in Fig. 1). DNA constructed plasmid is then used to obtain insertional plasmid pVE5.1, as described in example 1.

Example 4.

100-300 PCs 11-day-old chick embryos infect the fallopian m is CCW (HAO) embryo 0.1-0.2 ml vaccinated suspension, having a titer of 105106O. O. E./ml Infected embryos incubated 48 h at 37oC, then sterile open and separate areas HAO with drain defeat. The collected material is washed from inclusions in sterile Hanks solution, add 2 volumes of sterile 0.01 M buffer solution of Tris-HCl pH 9.0 and homogenized for 5 min in a mechanical homogenizer, cooling the mixture in an ice bath. The resulting suspension clarify by centrifugation (10 min, 750 g, 4oC), the resulting supernatant layer on 1/5 of the volume of the supernatant portion 36% solution of sucrose in 0.01 M Tris-HCl pH 9.0 and centrifuged (30 min, 23000 g, 4oC). All supernatant carefully removed, and the residue resuspended in 40 ml of 0.01 M solution of Tris-HCl pH 9.0 to homogenize for 1 min and 30 treated with ultrasound, and then repeat step centrifugation through a cushion of 36% sucrose solution. The precipitate malonebeach color suspended in sterile Hanks solution at a rate of 1 ml per 5 infected embryos, treated with 30 ultrasound, filled to 1 ml in a sterile microtube, frozen and stored until use at -40oC. To determine the titre of virus in the resulting material, typically having values of the new and purification of the virus is carried out in sterile conditions.

For long-term storage of the recombinant strain used storage technology natural strains of orthopoxviruses. The clarified homogenate suspension HAO infected chick embryos freeze-dried collector and drying unit for 22-24 hours Finished products are stored in ampoules, sealed under vacuum, at a temperature of minus (202oC.

Example 5.

Uterine material recombinant vaccinia virus, thoroughly checked for sterility and the presence of extraneous hemagglutinins impurities, with a specific title, injected intradermally in the 3-4 point of the shoulder region rabbits weighing 3-4 kg in the same way being re-vaccination after 28 days. The infectious dose of the recombinant virus S in both cases is 510710900E on each rabbit. 7 days after the second immunization each animal is injected subcutaneously with 100 LD50virus VAL. Observation of rabbits is still within 14 days. All rabbits, the past two immunization, stay alive and show no signs of disease, whereas all not vaccinated or vaccinated with the same dose of the original commercial strain of virus uspavano outbred white mice males weighing 7-10 g at a dose of 107O. O. E. mouse. Repeated immunization with the same dose of conduct for 21 days, and after 7 days the mice injected subcutaneously with 100 LD50virus VAL. In this experiment as a control were used to group net mice and mice that underwent the two - and single immunization with the same dose of the original virus strain S LIIT.

The results of the experiment are given in table. 1.

The vaccinated animals at various times after immunization with recombinant virus S take blood to determine the concentration of antibodies specific to VAL. To do this, use the following variant of solid-phase radioimmunoassay analysis: 300 ng of virus VAL in 10 μl of TBS buffer (0.01 M Tris-HCl pH 7.5; 0.1 M NaCl) containing 1% sodium dodecyl sulfate, put in wells of polystyrene plates and dried at 37oC, the wells washed with ethanol and incubated with 20 ml of RIA buffer (0.05 M Tris-HCl pH 7.5; 0.5 M LiCl; 0.15 M NaCl; 0.1% Triton X-100) containing 1% bovine serum albumin, and then 20 μl of RIA buffer containing 5-fold dilution of the serum (1 h at 37oC); after incubation, the wells are washed and contribute 20 µl of a solution of [125I] - labeled protein A (in each hole 2105pulse/min), incubated 1 h at 37oC and omavaltaisesti is performed on the RF. If analysis of mouse sera before binding of [125I]-labeled protein And the necessary additional incubation with rabbit serum against mouse IgG. Under the titer of the serum is understood as the maximum dilution at which the amount of bound peroxidase protein And 2 times background values.

The results are shown in table. 2.

From these data it follows that the two-time immunization of laboratory animals produced by a strain of recombinant vaccinia virus leads to the appearance in their blood high concentration of antibodies specific binding structural proteins VAL.

The present invention allows to obtain a recombinant strain of vaccinia virus that causes the synthesis of all the structural proteins VAL in infected cells, the accumulation of high concentrations of antibodies to the virus VAL in the body vaccinated with the recombinant vaccine animals and protect animals from lethal infection VAL.

The titer of antibodies specific to proteins VAL, in the serum of vaccinated twice in the same strain of animals is 1: 3000 to 1: 4000 for rabbits and 1: 625 1: 2000 for mice.

This concentration of specific immunoglobulins comparable with and in dozens of times higher than the concentration of antibodies in the blood of animals, immunized killed televizionnymi vaccines.

Such recombinant vaccinia virus can be the basis for the development of new generation vaccines against VAL and cooking immunological-based assays. Compared with attenuated strains of recombinant virus S may not have the ability reversion to pathogenic strain VAL, not reactogenic, easily produced and does not require to work with special precautions. Compared with the dead televizionnymi vaccines this strain has the following advantages: first, it does not need large-scale developments with subsequent isolation of the virus, which may significantly reduce the cost of vaccines; secondly, for killed vaccines do not exclude the possibility of incomplete inactivation of the active material, which leads to the development of the disease, live vaccines based on recombinant S such deficiencies have not.

The positive effect is achieved by installing a full-length DNA copies of the 26 S RNA encoding the structural proteins VAL under the control of the promoter of the gene of the protein of 7.5 To ospowiki in gene timedancing genome commercial strain ospowiki LIIT. The efficiency of gene expression, Waco genes for structural proteins of Genoa VAL, which leads to preservation of the right places initiation of protein synthesis predecessor order broadcast and transport of structural proteins VAL on the cell membrane, where they are efficiently presented to the immune system. High efficiency of transcription is provided by the use of natural strong promoter of the gene of the protein of 7.5 To vaccinia virus, it determines the high efficiency of the collected construction.

Thus, the proposed method of construction allows to obtain a recombinant strain of vaccinia virus, ensure the protection of laboratory animals from lethal infection called virus Venezuelan encephalomyelitis of horses.

LITERATURE

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11. Kinney, R. M. Esposito J. J. Johnson B. L. B. J. T. Roehrig Matheus J. H. Barrett, A. D. T. Trend D. W. (1988) Recombinant vaccinia/Venezuelan equine encephalitis (VEE) virus expresses VEE structural proteins. // J. gen. Virol. 1988. V. 69. N 12. P. 3005-3013.

12. Frolov, I. C. Kolykhalov A. A. Volchkov C. E. Netesov S. C., Sandakhchiev HP Comparison of amino acid sequences of the structural proteins attenuated and pathogenic strains of the virus Venezuelan encephalomyelitis of horses. // Reports of USSR Academy of Sciences. 1991. So 318. N 6. C. 1488-1491.

13. Venkatesan S. B. M. Baroudy Moss B. Distinctive nucleotide sequences adjacent to multiple initiation and termina N. N. Chizhikov C. E. Plasmid pTK1285 for introduction of foreign genes into the genome of vaccinia virus and the method of its construction. // The copyright certificate N 1640164. 8 December 1990

15. Maniatis T. Fritsch E., Sambrook J. Molecular cloning. Methods of genetic engineering. World: M. 1984. 480 C.

1. The recombinant strain of vaccinia virus GKV N 944, expressing the structural proteins of the virus Venezuelan encephalomyelitis of horses and suitable for the production of immunobiological preparations.

2. A method of obtaining a recombinant strain of vaccinia virus GKV N 944, namely, that from the original plasmid series pUC8 containing the DNA copy of the 26S-RNA virus Venezuelan encephalomyelitis of horses (WAL) length not less than 4000 p. N. having site recognition by the restriction enzyme EcoRI at the 3'-end and paired BamHI and SalGI sites at the 5'-end, remove SalGI-ApaI fragment, treated with plasmid DNA polymerase of phage T4 for the formation of blunt ends, recyclist DNA ligase of phage T4, transform the obtained plasmid cells of Escherichia coli and selected clones with plasmid containing DNA copy only 26S-RNA WEL without 5'-terminal sequence, then BamHI-EcoRI-fragment of this plasmid is combined with the DNA ligase of phage T4 fragment, RI, the obtained recombinant DNA, transform cells of Escherichia coli, selected clones with plasmid containing the DNA copy of the 26S-RNA WEL under the control of the promoter 7.5 K in the composition of the gene timedancing of vaccinia virus, allocate the indicated plasmid, which is used for recombination with the genome of vaccinia virus, and are selected by selection of a given strain.

 

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