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Recombinant canine influenza virus strain and preparation method thereof as well as vaccine prepared from recombinant canine influenza virus strain

A technology of canine influenza virus and avian influenza virus, applied in the field of bioengineering, can solve the problems of increasing human infection with H3N2CIV and lack of preventive vaccines, and achieve the effect of preventing or treating canine influenza and good immunogenicity

Inactive Publication Date: 2014-06-25
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

As a companion animal, dogs have very close contact with humans, which also increases the risk of human infection with H3N2CIV
[0004] Vaccines are an effective means to prevent and control viral infectious diseases, while H3N2CI is an emerging infectious disease in dogs. At present, there is no vaccine to prevent this disease at home and abroad

Method used

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  • Recombinant canine influenza virus strain and preparation method thereof as well as vaccine prepared from recombinant canine influenza virus strain
  • Recombinant canine influenza virus strain and preparation method thereof as well as vaccine prepared from recombinant canine influenza virus strain
  • Recombinant canine influenza virus strain and preparation method thereof as well as vaccine prepared from recombinant canine influenza virus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Rescue of recombinant CIV rH3N2-DL of the present invention

[0026] 1. RT-PCR amplification of HA and NA genes of H3N2 subtype canine influenza virus

[0027] Use the Axygen column virus RNA extraction kit to extract the H3N2 subtype canine influenza virus A / Canine / Liaoning / 27 / 2012 (H3N2) strain (abbreviated as CIV LN27, preserved by the Veterinary Microorganism Culture Collection Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) For RNA, see the kit instructions for specific steps. LN27cDNA 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 H2O19.0μL, LN27RNA5.0μL, AMV RT buffer8.0μL, 2.5mmol / L dNTP mixture4.0μL, Uni-12 universal primer 2.0μL, RNase Inhibitor1.0μL and AMVReverse Transcriptase1.0μL, total volume 40.0μL. Mix the above reverse transcription system, ...

Embodiment 2

[0043] Example 2 Identification of biological characteristics of recombinant CIV rH3N2-DL of the present invention

[0044] 1. Tissue culture half infection dose of recombinant CIV rH3N2-DL (50%tissue culture infectious dose, TCID 50 ) Determination:

[0045] The TCID of the 5th generation recombinant CIV rH3N2-DL was measured according to the WHO influenza operation manual 50 =10 4.8 / mL, while the TCID of the 5th generation CIV LN27 virus 50 =10 2.0 / mL, the replication titer of recombinant CIV rH3N2-DL on MDCK cells was more than 2log higher than that of parental strain CIV LN27.

[0046] 2. Chicken embryo half infection dose of recombinant CIV rH3N2-DL (50% egg infectious dose, EID 50 ) Determination:

[0047] The 5th generation CIV LN27 virus and the 5th generation recombinant CIV rH3N2-DL were made 10 times in sterilized 1×PBS respectively. 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 ...

Embodiment 3

[0052] Embodiment 3 Vaccine immune effect evaluation prepared by recombinant CIV of the present invention

[0053] Inoculate SPF chicken embryos or MDCK cells with recombinant CIV rH3N2-DL (preservation number: CGMCC No.8162), harvest chicken embryo allantoic fluid or cell culture supernatant, inactivate with 1‰ formaldehyde at 4°C for 72 hours, and inoculate through chicken embryos After the identification is completely inactivated, it is used as a vaccine antigen. The rH3N2-DL antigen is mixed with GEL A adjuvant and emulsified to make a vaccine. 1ml of vaccine (containing 8μg HA antigen) and 1ml of PBS were used to immunize 4 dogs through hindlimb muscles, and 1ml of PBS emulsified with adjuvant was used to vaccinate 4 dogs in the same way as a negative control. Booster immunization 3 weeks after the first immunization, the dosage method is the same as the immunization. 10 for 2 weeks after the second immunization 6 The virulent EID50H3N2 canine influenza virus (the viru...

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Abstract

The invention discloses a recombinant canine influenza virus strain and a preparation method thereof as well as a vaccine prepared from the recombinant canine influenza virus strain. A recombinant canine influenza virus comprises HA and NA genes of a H3N2 subtype canine influenza virus and six internal genes of a H9N2 avian influenza virus including PB2, PB1, PA, NP, M and NS. The recombinant canine influenza virus strain disclosed by the invention is named after rH3N2-DL. The preservation number of the recombinant canine influenza virus strain disclosed by the invention is CGMCC No.8162. The invention further discloses the preparation method of the recombinant canine influenza virus and the vaccine prepared from the recombinant canine influenza virus. Compared with the parental strain, the recombinant canine influenza virus disclosed by the invention has high virus titer and blood congeal titer both on chick embryos and MDCK cells. Animal experimental results show that the vaccine prepared from the recombinant canine influenza virus has good immunogenicity and protective efficacy.

Description

technical field [0001] The invention relates to a recombinant virus strain, in particular to a canine influenza recombinant virus, a preparation method thereof and a vaccine prepared therefrom. The invention belongs to the technical field of bioengineering. Background technique [0002] Canine influenza (Canine influenza, CI) is an acute and highly contagious respiratory disease of dogs caused by canine influenza virus (CIV) in the Orthomyxoviridae and Influenza virus type A influenza viruses. It is a fulminant epidemic and spreads very rapidly and extensively. [0003] The current epidemic of canine influenza is mainly caused by H3N8 subtype and H3N2 subtype canine influenza virus. H3N8 subtype canine influenza is caused by the cross-species transmission of equine influenza virus, which is mainly prevalent in foreign countries. There is no report of equine influenza virus infecting dogs in my country. Since 2006, incidents of avian H3N2 subtype influenza virus infecting ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/85A61K39/145A61P31/16C12R1/93
Inventor 刘明刘春国刘大飞张云
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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