Recombinant plasmid dna pc-ns3 integrating the complex of genes c, prm, e, ns1, ns2a, ns2b, ns3 virus tick-borne encephalitis (tick-borne encephalitis) into the genome of vaccinia virus (bob) and a recombinant strain of vaccinia virus expressing in cells of the immunized organism complex genes c, prm, e, ns1, ns2a, ns2b, ns3 tick-borne encephalitis virus

 

(57) Abstract:

The invention relates to the field of genetic engineering and biotechnology, in particular, to obtain the recombinant plasmid DNA RS-NS3 integrating the complex of genes C, prM, E, NSI, NS2A, NS2B, NS3 virus tick-borne encephalitis (tick-borne encephalitis) into the genome of vaccinia virus (WWII), and the corresponding strain of the great Patriotic war. The plasmid also contains the promoter P7, 5K BOB, synthetic DNA fragments with sites of initiation and termination of translation and DNA sequence of the gene timedancing WWII. The obtained plasmid DNA RS-NS3 used for the introduction of complex gene-NS3 tick-borne encephalitis in the genome of WWII. Recombinant BOB in the cells of the immunized organism enables the expression of complex antigens tick-borne encephalitis, which promotes the formation of protective immunity against infection with tick-borne encephalitis. 2 S. p. f-crystals, 1 tab., 1 Il.

The invention relates to biotechnology, in particular genetic engineering, and medicine, and is a recombinant strain of vaccinia virus (WWII), obtained using the methods of genetic engineering and designed for the development of a live vaccine against tick-borne encephalitis (TBE).

Tick-borne encephalitis - natural focal disease virus PEM is a virus tick-borne encephalitis (tick-borne encephalitis) from the kind of Flaviviruses. The virus is circulating in natural foci, human infection is through the bite of ticks, showing the greatest activity in the spring and summer.

An effective way of dealing with EC is vaccination. Used to this end, the preparations of inactivated vaccines has led to a significant reduction in the incidence of EC [1] . However arise when such vaccination immunity is short-term and requires repeated annual vaccination. Devoid of these shortcomings, live attenuated vaccine against TBE, but its application is suspended due to cases TBE resulting from vaccination [1].

In recent years, for the development of vaccines actively used the methodology of constructing recombinant viruses [2].

When you install it into the vaccinia virus part of the genome of the tick-borne encephalitis, encoding the major immunogenic proteins, you can expect to receive a live recombinant vaccines don't cause as complications of the disease EC. The use of vaccinia virus as a vector ensures the correct processing of the expressed antigens and the formation of a strong cellular immune response in vivo [3, 4] .

Vector ospowiki was widely used DL is suspended virulent strains. It was demonstrated protection of mice after immunization with recombinant BOB, expressing the genes of Dengue virus-4: E [5, 6], C-prM/M-E-NS1-NS2A [5], NS1-NS2A [7], as well as prM and E viruses Dengue-2 and Japanese encephalitis [8, 9].

The closest technical solution in this area is recombinant plasmid DNA p28 CME encoding the fusion polypeptide to obtain a live recombinant vaccine against tick-borne encephalitis [10, prototype, A. S. USSR N 1490963, CL C 12 N 15/00, 1993]. This plasmid construction contains a complex of genes VIK C, M and E, encoding all of the structural proteins of the virus except for the N-terminal amino acids of protein C and C-terminal part of the E protein under the control of the late promoter p28k of vaccinia virus. However, obtained by the proposed method, the recombinant vaccinia virus is not promising for the creation of an effective vaccine against TBE, because it has a weak protective properties compared to the control inactivated TBE vaccine.

A known strain of recombinant vaccinia virus V7 5c-producer crustal antigen of hepatitis B virus, providing the expression of C-gene of hepatitis B virus in mammalian cells [11, RF patent N 1788012, CL C 12 N 15/00, 1993]. In the patent and scientific-tekhnicheskaia.

It is known that gene expression VIK is through the synthesis of a single polypeptide, which is then cut into individual proteins by cellular proteases and protease viral nature [12].

It is known that the main immunogenum are the structural proteins prM/M (predecessor of membrane proteins and membrane protein), E - protein of viral envelope and non-structural proteins NS1 and NS3 [13]. In addition, it is shown the functional role of protein prM and virusspecific protease MS2B/NS3 in the Assembly and secretion of highly immunogenic E-containing virus-like particles [14, 15, 16] . Nonstructural protein NS2A, apparently, is involved in the processing of protein NS1 in a highly immunogenic form [7].

In addition, non-structural proteins NS3, NS1 or NS2A, NS4A or NS4B of Dengue viruses detected epitopes for T-helper cells and cytotoxic lymphocytes human and mouse[17, 18, 19, 20, 21].

Thus, existing data on immunological and functional role of nonstructural proteins NS2A, NS2B, NS3 of flaviviruses indicate the need for their inclusion in recombinant vaccine against tick-borne encephalitis.

An object of the invention is the creation of a recombinant strain of WWII, providing significant protective efficiency is due to the introduction of the target design, in addition to the structural genes (C, prM, E and non-structural NS1 gene, also complex non-structural genes NS2A-NS3.

The solution of the technical problem by constructing plasmid integration pC-NS3, providing the insert fragment cDNA tick-borne encephalitis, which encodes a polypeptide C-prM-E-NS1-NS2A-NS2B-NS3, into the genome of the great Patriotic war.

The plasmid consists of:

the cDNA fragment of tick-borne encephalitis strain sofjin;

a synthetic DNA fragment containing the site of translation termination;

promoter BOB;

plasmid vector that provides the insertions of foreign genes in gene timedancing (tk gene), BOB.

As a fragment of the cDNA tick-borne encephalitis is a fragment of DNA 6322 p. N. , involving complex gene C (except for the first three codons.K.), prM, E, NS1, NS2A, NS2B, NS3.

As a site of translation initiation, we use a synthetic DNA fragment length of 21 p. N. having the structure:

< / BR>
This synthetic fragment includes the codons of the first three amino acids of the gene C.

As a promoter BOB used ClaI-BamHI-fragment DNA plasmids pVAR-15 length 270 p. N., containing the early-late promoter BOB p7.5k [22].

As a site of translation termination, we use a synthetic DNA fragment length is in the tk-gene, BOB, used plasmid pTK1614 [23].

The obtained plasmid integration pC-NS3 used for the introduction of complex genes C-NS3 VIK under the control of a promoter BOB p7.5k in the genome of the BOB strain LIPS in the process of cotransfection plasmid and viral DNA of culture cells CV-1. The result of homologous recombination between the DNA plasmid integration and BOB received a recombinant strain of WWII kv26, the genome consists of genes C, prM, E, NS1, NS2A, NS2B, NS3 VIK.

The essence of the invention lies in the fact that the expression of complex genes C-NS3 tick-borne encephalitis in the composition of recombinant vaccinia virus kv26 in the cells of the immunized organism determines the synthesis of antigens tick-borne encephalitis, causing the formation of protective immunity against infection with virulent strain of tick-borne encephalitis. A significant protective effect is achieved by introduction of a recombinant strain of WWII optimal set of genes VIK.

Morphology, signs of strain

Recombinant strain kv26 has properties typical representation of a family of orthopoxviruses. Virions strain kv26 have a size 200x300 nm, characteristic biketours the form and according to electron microscopy does not differ from the original strain LIVP.

Cultural properties ø the th action and lysis of infected cells. Unlike strain LIVP has TK--phenotype - reproducing cells of Rat-2(TK-in the presence of 25 μg/ml of bromosuccinimide. While infecting a 12-day-old developing chick embryos (RCA) from chickens breed Livorno in doses of 101- 102opinionbased units (AOE) causes the formation of gorillatorch membranes (HAO) of white solid lesions (raised bumps) with a diameter of from 0.5 to 3 mm for cutaneous infection of rabbits and calves causes typical vaccination lesions.

Virulent and toxicogenic properties

Strain kv26 harmless in the test on Guinea pigs and outbred mice. When intracerebral administration to rabbits at a dose of 5104OOE and mice at a dose of 1104AOE does not cause death. When intradermal administration to rabbits at a dose of 5105AOE does not cause the formation of necrosis.

DNA strain kv26 has a length of about 180 thousand p. N., and the place HindIIIK-fragment (5000 p. N.) strain LIVP appear three new HindIII fragment length 2800 p. N., 4250 p. 4575 N. and p. N. Fragments 2800 p. 4575 N. and p. N. according to DNA-DNA blot hybridization contain cDNA VIK.

The main difference of strain kv26 from LIIT is the ability to protect mice during infection with tick-borne encephalitis. High degree the second immunization virus kv26 and considerably exceeded the level of protection of mice during infection with tick-borne encephalitis following immunization with strain-prototype wr7.5CMENS1 in similar conditions.

The strain described recombinant strain of WWII kv26 deposited in the Public collections of viral strains Institute of Virology. D. I. Ivanovsky number GKV N 945.

Thus, for the first time obtained a recombinant strain of the great Patriotic war, expressing complex genes C-NS3 tick-borne encephalitis and determining the synthesis of antigens VIK, providing a significant protective effect against TBE.

The invention is illustrated in the following graphics: drawing given scheme plasmid integration pC-NS3 and method of construction.

For a better understanding of the essence of the present invention the following are examples of its implementation.

Example 1. Method of constructing plasmid integration pC-NS3. 50 μg DNA plasmid p10+4 with the cloned cDNA fragment VIK length 2100 p. N., encoding structural proteins of tick-borne encephalitis, split BglI-restriction enzyme under standard conditions [24, 25]. BglI(n.)-BglI(n.)-the cDNA fragment VIK length 1074 p. N. , genes including C, prM(M) and the beginning of the gene E VIK, isolated by electrophoresis in agarose gel with subsequent Electrosila on dialysis membrane [24]. Numbering of the nucleotide residues of the genome of the tick-borne encephalitis is provided in accordance with the data Pletnev et al. [24].

In addition, p is s conditions with vector and a synthetic DNA fragment, including the site of translation initiation and having the following structure [25]:

< / BR>
As the vector is a plasmid pVAR-15, hydrolyzed by BamHI and EcoRI sites. Ligase mixture transform competent cells of E. coli HB101. Desired clones determined by the appearance of DNA fragment length 1800 p. N. when the joint hydrolysis of restrictase BamHi and EcoRI. The obtained plasmid, named pCME(316), then used in the construction of plasmids pC-NS1.

To obtain plasmid constructions pC-NS1 used clones provided Pletnev, A. G. (NIVKH, SB) p10+4, p18 [23]. Clones are the following fragments:

1. EcoRI(n. )-HinfI(n. )-fragment length 217 p. N. from plasmids p10+4.

2. HinfI(n.)-HinfI(n.)-fragment length 252 p. N. from plasmid p18.

3. HinfI(n.)-FokI(n.)-fragment length 114 p. N. from plasmid p18.

Selections, including the 3'end of the gene E and 5'-end of the NS1 gene, are ligated with FokI-hydrolysate SalGI-EcoRI fragment isolated from the plasmid pBR327NS1' and contains the sequence of the NS1 gene [26]. When this happens SalGI-FokI(n.)-district SalGI-EcoRI fragment length 47 p. N. EcoRI fragment(n. )-FokI(n. ) length 583 p. N., and restore the integrity of the remaining sequence SalGI-EcoRI fragment.

the d transform competent cells of E. coli HB101. Desired clones are selected based on the presence of EcoRI(1914)-EcoRI fragment length 1783 p. N. proper Assembly and orientation determined by the occurrence of fragments of length p. 1986 2991 N. and p. N. by the hydrolysis of BglI-restrictases.

The obtained plasmid pC-NS1 used when designing the target plasmid pC-NS3.

As a source of non-structural genes NS2A, NS2B and NS3 use plasmids pBR-NS2 and pGEM2-NS3 [27].

To obtain plasmid constructs pBR-NS2 uses clones provided Pletnev, A. G. (NIVKH MORAN) p8, p15 and p6 [24]. Clones are the following fragments:

1. HindIII(n. )-NcoI(n.)-fragment length 513 p. N. and NcoI(n. )-HhaI(n.)-fragment length 105 p. N. from plasmids p8.

2. HhaI(n.)-SacI(n.)-fragment length 624 p. N. from plasmids p15.

3. SacI(n.)-SalGI(n.)-fragment (203 p. N.) from plasmid p6.

The fragments are ligated under standard conditions with a vector plasmid pBR 322, hydrolyzed by HindIII and SalGI sites. Ligase mixture transform competent cells of E. coli HB101. Desired clones determined by the appearance of fragment length 1445 p. N. in joint HindIII-SalGI-hydrolysate of plasmid DNA.

From the resulting plasmid pBR-NS2 allocate HindIII fragment(n.)-SalGI(n.) length 1446 p. N., containing the sequence encodergasm 3'-terminal part of NS3 gene and a synthetic DNA fragment with a length of 16 p. N., including website translation termination and having a structure:

< / BR>
Ligase mixture hydrolyzing their endonuclease HindIII restriction and produce HindIII(3673 p. N.)-HindIII fragment length 2801 p. N., which is used as a source of complex genes NS2A-NS3.

To obtain the target plasmid pC-NS3 plasmid DNA pC-NS1 hydrolyzing ClaI, HindIII-restrictable. The selected fragment length 3850 p. N., containing the promoter p7.5k and genes C, M, E, NS1, are ligated with the vector pTK1614, hydrolyzed by ClaI and HindIII sites. Ligase mixture transform competent cells of E. coli HB101. Desired clones determined by the appearance of DNA fragment length 3850 p. N. when the joint hydrolysis ClaI and HindIII by restrictase. The obtained plasmid pTKC-NS1 hydrolyzing the restriction enzyme HindIII and are ligated with HindIII(n.)-HindIII fragment carrying the genes NS2A, NS2B, NS3 and website translation termination. Ligase mixture transform competent cells of E. coli HB101. Desired clones determined by the occurrence of fragments of length 3950, and 2800 3850 p. N. in joint Cla-HindIII-hydrolysate of plasmid DNA. Thus obtained plasmid denoted pC-NS3 and used for integration of complex genes C-NS3 gene of WWII when receiving recombinant strain kv26.

Example 2. The preparation of recombinant VI is the accepted method of cotransfected plasmid and viral DNA of culture cells CV-1, infected BOB strain LIVP [28]. The building complex of genes C-NS3 VIK spend HindIIIK-DNA fragment of WWII. Culture vessels with monolayer cells CV-1 (20 cm2) infect BOB with a plurality of 0.05-0.1 VOYE/CL Adsorption of the virus on the cells is carried out for 1 h at 37oC, then remove neadsorbirovanne virus and cells are covered by a supporting environment the Needle MEM containing 2% fetal serum and 80 u/ml of gentamicin. After 1 h incubation at 37oC a supportive environment is drained and the cells put 0.5 ml of calcium-phosphate precipitate containing 1 μg of DNA plasmid integration and 20 μg DNA of WWII, and incubated at room temperature. After 45 min after application of the precipitate cells cover of 4.5 ml of eagle medium MEM containing 8% fetal serum and 80 IU/ml gentamicin and incubated for 18-24 hours in the Cells after transfection frozen, thawed and clone viral progeny cells of transplantable culture Rat-2(TK-under agar coating containing 25 μg/ml VDU. After 48 h incubation at 37oC in an atmosphere containing 5% CO2cause the second floor, containing 0.01% dye neutral red. Staining of monolayer cells for visualization of viral plaques spend recerived in 24-hole plastic cards (the diameter of the hole 16 mm) and analyzed using DNA-DNA dot-hybridization with labeled-P32the cDNA fragment VIK on the availability of cDNA sequences VIK.

Selected using the dot-hybridization clone recombinant BOB subjected to repeated cloning. A suspension of the virus is treated with ultrasound for 1 min, filtered through a filter with pore diameter of 0.45 μm and retitrement on cells of Rat-2(TL-under agar coating containing 50 μg/ml VDU, as described above. Individual viral plaques select and develop virus HAO RCA.

Of the purified recombinant virus secrete DNA [28, 29] and examine it using restriction analysis. Conducted by restriction analysis showed that the cDNA insertions tick-borne encephalitis occurred in HindIIIK-fragment (5000 p. N.) genome BOB (strain LIVRE), as evidenced by the disappearance of this fragment in the hydrolyzed DNA recombinant virus and the emergence of new HindIIIK fragments length 2800 p. N. , 4250 p. 4575 N. and p. N. On the correctness of the installation is also evidenced by the coincidence of the electrophoretic mobility of fragments of length 2800 p. 3368 N. and p. N. accordingly, in HindIII and > PST - hydrolysates of DNA plasmids pC-NS3 and recombinant strain.

The obtained recombinant strain of WWII was marked as kv26, lyophilized and deposited in the State the activity of the recombinant strain kv26.

As an experimental model using mice of Balb/c 10-12 g weight. Immunization strain LIVP and recombinant strains kv26 and wr7.5CMENS1 carried out by the method of cutaneous scarification of the base of the tail of mice. The drug is applied in a volume of 5 μl, which is 107OOE.

As a positive control take the mice immunized with inactivated culture TBE vaccine production TOMMIES intraperitoneally at 0.1 ml per mouse.

As virulent test strain of tick-borne encephalitis take the strain Absettarov used for 30 years for laboratory evaluation in mice immunogenic vaccines against tick-borne encephalitis.

Infection of mice VIK spend intraperitoneally tenfold dilutions of virus (from 2.0 to 7.6 lg LD50) at a dose of 0.1 ml 21 days after immunization. Each dilution of virus infect 8-10 mice. As a control test strain used intact mice of the same age, which is titrated virus. Dead animals are recorded daily for 2 weeks after infection. The mouse that died in the first 3 days are not counted. LgKD50calculated by the method of reed and turns into a hissing drone [31]. Protectively determined by the resistance index (RI). RI is calculated by the behavior of animals after immunization were observed. Specific killing of the animals after administration of the test strains of TBE virus was noted on 6 days, which corresponds to a known term incubation of tick-borne encephalitis virus.

The results determine the protective properties of recombinant strains of BOB expressed in LD50and RI for each group of immunized mice presented in the table. The resistance index for mice immunized with strain kv26 was 7.0, which means that the animals were completely protected against infection with tick-borne encephalitis in the dose of 107LD50. The level of protectively comparable with the level of protection after immunization commercial inactivated vaccine EC and significantly higher than after immunization with strain-prototype wr7.5CMENS1 (RI= 4.0).

Thus, for the first time obtained a recombinant strain of the great Patriotic war, expressing the complex C-NS3 tick-borne encephalitis and determining the synthesis of antigens VIK, providing a significant protective effect against TBE.

Literature

1. Smorodintsev A. A., Oaks, A. C. Tick-borne encephalitis and its prevention. Leningrad: Medicine, 1986.

2. Mackett, M., Smith, G. L. J. Gen. Virol., 1986, v. 67, pp. 2067-2082.

3. Moss B., Sciens, 1991, v. 252, pp. 1662-1667.

4. Moss B., In: Seminars in Virology, Vol. 3, Academic Press Ltd., 1992, pp. 277-283.

., J. Virol., 1990, v. 64, pp. 4356-4363.

8. Schlesinger, J. J., Putnak, J. R. and K. H. Eckels In: Vaccines: New Approaches to Immunological Problems. R. W. Ellis, ed., Butterworth-Heinemann, Boston, MA., 1992, pp. 289-307.

9. Konishi, E., et al., Virology, 1992, v. 190, pp. 454-458.

10. Auth.St. USSR N 1490963, CL C 12 N 15/00, BI N 25, 1993

11. RF patent 1788012, CL C 12 N 15/00, 1993

12. Rice C. M. , Strause E. G. and J. H. Strause In: The Togaviridae and Flaviviridae. Schlesinger S. and Schlesinger, M. J., eds., Plenum Press, New York, 1986, pp. 279-327.

13. R. Putnak, In: Modern Vaccinology. E. Kurstak, ed., Plenum Medical, New York, 1994, pp. 231-252.

14. Konishi, E., Virology, 1992, v. 188, pp. 714-720.

15. Sato T. et al., Virology, 1993, v. 192, pp. 283-490.

16. Yamshchicov V. F. and Compans, R. W., Virology, 1993, v. 192, pp. 38-51.

17. Kurane I. et al., J. Clin. Invest., 1989, v. 83, pp. 506-513.

18. Kurane I. et al., J. Virol., 1991, v. 65, pp. 1823-1828.

19. Bukowski, J. F. et al., J. Virol., 1989, v. 63, pp. 5086-5091.

20. Rothman, A. L. et al., J. Virol., 1989, v. 63, pp. 2486-2491.

21. Rothman, A. L. et al., J. Virol., 1993, v. 67, pp. 801-806.

22. Fodor, I. I. and others, Biotechnology. 1987, so 3, N 3, S. 302 to 306.

23. Prikhod'ko, G. Structural and functional studies of genes of vaccinia virus strain LIVP. Dessert. for obtaining the academic degree of candidate of Biol. Sciences in the form of scientific. report, Novosibirsk, 1990.

24. Pletnev, A. S., Yamshchikov, V. F., V. M. Blinov, Virology, 1990, v. 174, No. 1, pp. 250-263.

25. Maniatis T. and other Molecular cloning. M: Mi, . 297, N 1, 2, pp. 67-69.

28. Smith, G. L., Mackett, M., Moss B., Nature, 1983, 302 N, pp. 409-495.

29. Nakano E. , Panicali P., Paoletti E., Proc. Natl. Acad. Sci. USA, 1982, v. 79, pp. 1533-1536.

30. Joklick W. K., Virology, 1962, v. 18, No. 1, pp. 9-18.

31. Reed, L. J. and Muench, H., Amer. J. Hyg., 1938, v. 37, pp. 493-497.

1. Recombinant plasmid DNA pC-NS3 integrating the complex of genes C, prM, E, NS 1, NS 2A, NS 2B, 3 NS of tick-borne encephalitis virus (VIK) into the genome of vaccinia virus (WWII), mol.m. 6,9 MDa and size 10600 N. p., containing

Bg l I-Alu I fragment of cDNA VIK length 6322 p. N., encoding complex C genes (with the exception of the first three codons of amino acids), pr, M, E, NS 1, NS 2A, NS 2B, 3 NS;

Bam H I-Bg l I - a synthetic DNA fragment (21 p. N.), having the structure

,

encoding the effective site of translation initiation and the first three amino acids of polyprotein tick-borne encephalitis;

Bam H I-Hind III - synthetic DNA fragment (16 p. N.), having the structure

< / BR>
coding website translation termination;

Cla I-BamH I fragment of DNA plasma pVAR-15 length 270 p. N., containing the early-late promoter BOB p.7.5;

plasmid vector RTC 14, linearized by site Cla I, Hind III and containing the gene for resistance to ampicillin and the site of initiation of replication of plasmids pBR322, and the nucleotide sequence of the gene timedancing Boost to ampillicin transformation of E. coli;

t - interrupted gene timedancing BOB, causing TC-the phenotype by transformation TC+BOB;

unique sites for endonucleases of restriktsii:

Cla I site between the sequence of a gene timedancing and 7.5 sequence to a promoter;

Xba I site between the cDNA sequence of tick-borne encephalitis and sequence of the gene timedancing.

2. Recombinant strain of vaccinia virus GKV N 945 expressing in cells of the immunized organism complex genes C, prM, E, NS 1, NS 2A, NS 2B, 3 NS virus adhesive encephalitis.

 

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