Recombinant European avian-borne H1N1 subtype swine influenza vaccine strain, and preparation method and application thereof

A technology of recombinant vaccine and swine flu, which is applied in the field of bioengineering, can solve problems such as hindering the development and use of influenza virus and low adaptability of influenza virus, and achieve the effect of high antibody titer, high virus titer, and complete and effective immune protection

Active Publication Date: 2017-02-08
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, influenza viruses generally have low adaptability on common MDCK cells, which hinders their further development and use.

Method used

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  • Recombinant European avian-borne H1N1 subtype swine influenza vaccine strain, and preparation method and application thereof
  • Recombinant European avian-borne H1N1 subtype swine influenza vaccine strain, and preparation method and application thereof
  • Recombinant European avian-borne H1N1 subtype swine influenza vaccine strain, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of European Avian H1N1 Subtype SIV Recombinant Strain SH / PR8

[0045]According to the instructions on the TRizol kit, the viral RNA of the domestic epidemic strain A / swine / Shanghai / 1 / 2014 (SH1) of European poultry-origin H1N1 subtype swine influenza virus was extracted, and cDNA was synthesized by reverse transcription, and the purpose was carried out by RT-PCR. Amplification of fragment HA and NA, PCR reaction conditions: 95°C, 1min; 95°C, 20s; 48°C, 20s; 72°C, 1min; 72°C, 10min, 30 cycles. The amplified HA and NA nucleic acid products were identified by 1% agarose gel electrophoresis (see figure 1 , figure 1 Middle, M: DNA molecular mass standard (DL2000); 1: PCR amplification product of NA gene (about 1410bp); 2: PCR amplification product of HA gene (about 1701bp)), and the product was recovered. Respectively, the gel recovery products containing HA and NA were digested by the restriction endonuclease BspQI, and then gel recovered, and then c...

Embodiment 2

[0046] The comparison of embodiment 2 recombinant strain SH / PR8 and original wild strain SH1 on cell and chicken embryo replication ability

[0047] Prepare influenza virus infection liquid, add TPCK (protease). Dilute the virus, according to the 10-fold dilution method, dilute the virus with the virus infection solution, mix it well, and dilute the virus from 10 -1 diluted to 10 -11 , add the diluted virus to the 96-well plate lined with MDCK cells in sequence, and put it in 37°C, 5% CO 2 Culture in the cell incubator for 72 hours, observe the cell pathological changes (CPE), and record the results, aspirate the cell supernatant for the hemagglutination test to check the test results, and calculate the TCID 50 value.

[0048] Prepare influenza virus infection solution, dilute SH / P8 and original strain SH1 virus with virus infection solution, and inoculate aseptically into 12-well cell culture plate according to the virus amount of 0.001MOI per well, and the 12-well cell cu...

Embodiment 3

[0051] Example 3 Preparation of European poultry-derived H1N1 subtype SIV recombinant high-yield inactivated vaccine and evaluation of its immune protection efficacy

[0052] The original wild strain SH1 and the recombinant strain SH / PR8 were inoculated into MDCK cell culture flasks at a dose of 0.001 MOI. After a large number of viruses were amplified, the cell supernatant was collected, and the cell debris was separated by centrifugation at 3000rpm for 10min and the supernatant was collected. The HA hemagglutination test was performed on the amplified original wild strain SH1 and the recombinant virus SH / PR8 to detect the virus hemagglutination titer.

[0053] The Montanide ISA 61VG water-in-oil mineral adjuvant and Montanide ISA 15AVG oil-in-water mineral adjuvant produced by Sepic Company of France were selected for comparison. The virus was inactivated by 0.1% formaldehyde solution at 37°C for 24-48 hours, and the inactivated virus was blindly passed on MDCK cells for 3 g...

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Abstract

The invention discloses a recombinant European avian-borne H1N1 subtype swine influenza vaccine strain. The recombinant vaccine strain comprises the HA and NA genes of an European avian-borne H1N1 subtype swine influenza virus strain SH1 and six inner genes consisting of PA, PB1, PB2, M, NP and NS of a PR8 virus. The invention also discloses a preparation method and application thereof of the recombinant swine influenza vaccine strain. The recombinant swine influenza vaccine strain has high virus titer on MDCK cells; and a recombinant high-yield inactivated vaccine prepared through inactivation of the recombinant swine influenza vaccine strain can provide complete-response immunoprotection against attack by European and Asian avian-borne H1N1 subtype swine influenza viruses.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant European poultry-derived H1N1 subtype swine influenza vaccine strain and a preparation method and application thereof. Background technique [0002] Swine influenza (SI) is a highly contagious respiratory infectious disease caused by swine influenza virus (Swineinfluenza virus, SIV) of the family Orthomyxoviridae, which causes symptoms such as high fever, cough, and dyspnea. It has the characteristics of high morbidity, rapid transmission and low mortality. It has been reported that swine influenza virus infects pigs and can easily cause mixed infections of other bacteria and viruses, such as porcine reproductive and respiratory syndrome, infectious pleuropneumonia, Streptococcus suis, etc., making the epidemic more complicated and affecting the disease. Control poses great difficulties. At present, the SIV subtypes that cause pig disease are mainly H1N1, H...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85A61K39/145A61P31/16C12R1/93
Inventor 于海童光志阮宝阳宫晓倩李泽君汪秀会刘晓敏
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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