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Recombinant equine influenza virus strain, preparation method thereof and vaccine prepared from recombinant equine influenza virus strain

A technology of influenza virus and equine influenza, applied in the field of bioengineering, can solve the problems of high price, low antigen specificity, and inability to provide complete protection

Inactive Publication Date: 2014-02-26
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sequence analysis of the H3N8 subtype EIV isolated in my country showed that the EIV popular in my country belongs to the American lineage Florida 2 group, while the American lineage vaccine strain corresponding to the vaccine Proteq-Flu1 used in my country belongs to the Kentucky sub-lineage. There are 15 amino acid mutations on the HA gene, and 8 of them are located on the A, B and C antigenic regions respectively. Protect
Due to the high price of imported vaccines and the shortcomings of antigenicity, it is of great significance to develop a new type of high-efficiency EI vaccine with independent intellectual property rights in my country and good protection.

Method used

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  • Recombinant equine influenza virus strain, preparation method thereof and vaccine prepared from recombinant equine influenza virus strain
  • Recombinant equine influenza virus strain, preparation method thereof and vaccine prepared from recombinant equine influenza virus strain
  • Recombinant equine influenza virus strain, preparation method thereof and vaccine prepared from recombinant equine influenza virus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Rescue of recombinant EIV rH3N8 of the present invention

[0031] 1. RT-PCR amplification of EIV XJ3HA and NA genes

[0032] Use the Huashun column virus RNA extraction kit to extract the RNA of equine influenza virus A / equine / xinjiang / 3 / 07 (H3N8) (referred to as XJ3 virus). For specific steps, refer to the kit instruction manual. XJ3 cDNA was prepared by using the universal primer for reverse transcription of influenza A virus Uni-12: 5'-AGCAAAAGCAGG-3' as the reverse transcription primer. The reverse transcription system was as follows: DEPC H2O 19.0 μL, XJ3 RNA 5.0 μL, AMV RT buffer 8.0 μL, 2.5mmol / L dNTP mixture4.0μL, Uni-12 universal primer 2.0μL, RNase Inhibitor1.0μL and AMV Reverse Transcriptase1.0μL, total volume 40.0μL. Mix the above reverse transcription system, let it stand at room temperature for 10 minutes, put it in a water bath at 42°C for reverse transcription for 1 hour, then place the reverse transcription product at -4°C for 2 minutes, and ...

Embodiment 2

[0048] Example 2 Identification of biological characteristics of recombinant EIVrH3N8-PR of the present invention

[0049] 1. Tissue culture half infectious dose (50% tissue culture infectious dose, TCID) of recombinant EIV rH3N8-PR 50 ) Determination:

[0050] According to the WHO influenza operation manual, the TCID of the 5th generation recombinant EIV rH3N8-PR was measured 50 =10 5.2 / ml, while the TCID of the 8th generation XJ3 virus 50 =10 2.2 / ml, the replication titer of recombinant EIV rH3N8-PR on MDCK cells was 100 times higher than that of XJ3.

[0051] 2. Half infection dose of chicken embryos of recombinant EIV rH3N8-PR (50% egg infectious dose, EID 50 ) Determination:

[0052] Make 10 copies of 8th generation XJ3 virus and 5th generation recombinant EIV rH3N8-PR with sterilized 1×PBS 5 、10 6 、10 7 、10 8 、10 9 and 10 10 For each dilution, 5 SPF chicken embryos were inoculated, 0.2 mL per embryo, placed in a 37°C incubator for 72 hours, and the allantoi...

Embodiment 3

[0061] Example 3 Vaccine immunogenicity experiment prepared by recombinant EIV of the present invention

[0062] Inoculate SPF chicken embryos or MDCK cells with recombinant equine influenza virus rH3N8-PR, harvest chicken embryo allantoic fluid or cell culture supernatant, inactivate with 1‰ formaldehyde at 4°C for 72 hours, and inactivate completely after chicken embryo inoculation Purified by sucrose density gradient centrifugation as vaccine antigen. The content of HA protein was measured, and the rH3N8-PR antigen containing 15 μg, 30 μg and 50 μg of HA protein was mixed with GEL A adjuvant and emulsified to make a vaccine. Three different doses of the vaccine were used to immunize 2 horses via the neck muscle respectively. Booster immunization 4 weeks after the first immunization, the dosage method is the same as the immunization. Blood was collected 1 day before immunization and every week after immunization, the serum was separated, and the titer of HI antibody was de...

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Abstract

The invention discloses a recombinant equine influenza virus strain, a preparation method thereof and a vaccine prepared from the recombinant equine influenza virus strain. The recombinant influenza virus strain contains genes HA and NA of an equine influenza virus A / equine / xinjiang / 3 / 07 (H3N8) strain and six internal genes PB2, PB1, PA, NP, M and NS of an influenza virus A / Puerto Rico / 8 / 34 / Mount Sinai (H1N1) or A / PR / 8 / 34 (H1N1 short for PR8 virus). The recombinant equine influenza virus strain disclosed by the invention is named as rH3N8-PR and is preserved with the number of CGMCC NO.8161. The invention also discloses a preparation method of the recombinant equine influenza virus strain and a vaccine prepared from the recombinant equine influenza virus strain. Compared with a parental strain, the recombinant equine influenza virus strain disclosed by the invention can generate very high virus titer and blood clotting titer on both chick embryos and MDCK (Madin Darby Canine Kidney) cells, and the pathogenicity of the recombinant equine influenza virus strain to mice is remarkably reduced; experiments prove that the vaccine prepared from the recombinant equine influenza virus strain disclosed by the invention has favorable immunogenicity and protective effect.

Description

technical field [0001] The invention relates to a recombinant virus strain, in particular to a recombinant equine influenza virus, a preparation method thereof and a vaccine prepared therefrom. The invention belongs to the technical field of bioengineering, Background technique [0002] Equine influenza (Equine influenza, EI) is caused by equine influenza virus (Equine influenza virus, EIV) in the Orthomyxoviridae, Influenzavirus type A influenza virus, a severe common upper respiratory acute high-level exposure of equine animals Sexually transmitted diseases are mostly explosive and spread very quickly and widely. OIE stipulates that EI is a legally notifiable animal disease, and my country lists it as a third-class infectious disease. [0003] The currently popular EI is mainly the H3N8 subtype. However, due to various pressures, the H3N8 subtype EIV is also constantly mutating, resulting in the emergence of multiple lineage viruses, and EIV has crossed the interspecies ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/63A61K35/76A61K39/145A61P31/16C12R1/93
Inventor 刘明刘春国相文华刘大飞张云曲连东
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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