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A kind of recombinant virus for preventing viral myocarditis and its vaccine and application
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A technology of recombinant virus and stomatitis virus, applied in the direction of antiviral agent, virus/bacteriophage, recombinant DNA technology, etc., can solve the problem of poor effect of viral myocarditis, achieve prevention of viral myocarditis, enhance induction, and good virality The effect of myocarditis
Active Publication Date: 2018-02-23
SUZHOU UNIV
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Problems solved by technology
[0007] The object of the present invention is to provide a kind of recombinant virus for preventing viral myocarditis and its vaccine and application, and described this kind of recombinant virus for preventing viral myocarditis and its vaccine and application have solved the problems in the prior art The technical problem that the vaccine is not effective against viral myocarditis caused by Coxsackievirus type B3 (CVB3) infection
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Embodiment 1
[0041] Example 1 Hela cell culture and CVB3 virus passage:
[0042] The human cervical cancer cell line Hela cell of the present invention is cultivated according to a conventional method, with RPMI-1640 medium containing 10% NBS, 2mM L-glutamine, 100U / ml penicillin and kanamycin sulfate at 37°C, 5% CO 2 cultured under the same conditions and passaged every other day. Infect about 5×10 6 Hela cells, after 40 hours of culture, 80% of the cells were lysed by the replicating virus, and the liquid and cell debris were centrifuged at 3000 rpm / min for 20 minutes, and the obtained supernatant was fresh CVB3 suspension.
Embodiment 2
[0043] LD of embodiment 2 CVB3 virus 50 (LD50) titration:
[0044] BALB / c suckling mice born for 48 hours were taken out and divided into 8 groups with 6 mice in each group. -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 , respectively take 100 μl and inject 8 groups of suckling mice through intraperitoneal cavity, observe the survival situation of suckling mice every day, and calculate the LD of this batch of CVB3 virus according to the routine method of virology 50 potency. Distance ratio = (>50% death percentage - 50) / (>50% death percentage - 50 => 50% of the logarithm of the mortality dilution (Log) + distance ratio
[0045] LD of CVB3 in the same batch in this test 50 Valence is 10 -5.5 / 100ul.
Embodiment 3
[0046] Example 3 Construction of recombinant VSV virus and identification of expression function in vitro
[0047] 1 Construction of the target antigen plasmid pXN2-VP1: the pXN2-VP1 plasmid with pXN2 as the carrier was constructed with the CVB3 structural protein VP1 as the target antigen. Wherein, the VP1 cDNA sequence is based on literature (Klump WM, et al. J Virol, 1990, 64(4):1573-1583), specifically shown in SEQ ID NO:1.
[0048] 1) The CVB3VP1 gene was obtained from freshly cultured CVB3 (Nancy strain) by RT-PCR.
[0049] Design the upstream and downstream primers of VP1: KV1: 5′-CCCAAGCTTGCCACCATGGGCCCAGTG GAAGACGCG-3′; KV2: 5′-CGGGATCCTTACTAAAATGCGCCCGTA TTTGT C-3′. Viral RNA was extracted according to the RNAexReagent&System Kit of Shanghai Huashun Biological Company, and the total RNA of CVB3 was mixed with 20 μl DEPC H 2 dissolved in O and stored at -80°C.
[0050] The reverse transcription of cDNA was carried out according to the 2-N First Strand cDNA Synthesi...
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Abstract
The invention relates to a recombinant virus, which uses vesicular stomatitis virus as a carrier, and inserts a gene encoding antigen CVB3 structural protein VP1 between envelope glycoprotein G and polymerase protein L of vesicular stomatitis virus. The present invention also provides a method for preparing the above-mentioned recombinant virus, which is obtained by transfecting BHK21 cells with the five plasmids pP, pL, pXN2‑VP1, pVSVG and pN. The present invention also provides a vaccine containing an effective amount of the above-mentioned recombinant virus. The vaccine of the present invention can be vaccinated in mucosal parts by nasal drops, oral administration or through the reproductive tract, which can solve the problem that conventional vaccines in the prior art cannot effectively induce a high-intensity mucosal immune response, and the antigen cannot effectively stay in the local mucosa and is not enough to be absorbed by APC. technical questions raised. The recombinant virus of the present invention can effectively enhance CVB3-specific serum and local mucosal antibody responses, and significantly enhance the specific CD8 T cell killing ability of the whole body and intestinal mucosa.
Description
Technical field: [0001] The invention relates to biological gene technology, in particular to a recombinant virus, in particular to a recombinant virus for preventing viral myocarditis and its vaccine and application. Background technique [0002] Viral myocarditis is mainly caused by Coxsackie virus type B3 (CVB3) infection, and the incidence is higher in young adults, which can cause about 50% of human acute and chronic myocarditis and 25% of dilated cardiomyopathy. It is a common and frequently occurring disease. diseases of the cardiovascular system. There is currently no effective preventive or therapeutic vaccine. CVB3 belongs to enterovirus. After invading the body through the respiratory tract and gastrointestinal tract, the body's mucosal immunity (such as intestinal IgA) cannot clear the virus in the early stage of infection, so the virus quickly spreads into the blood and then invades the heart, resulting in viral myocarditis; Persistent infection of CVB3 may al...
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