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A technology of influenza virus and strain, applied in the field of preparation of influenza virus vaccine strain, can solve the problems of inconvenience and low virus growth titer
Active Publication Date: 2017-02-08
CHINA AGRI UNIV
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Therefore, an ideal vaccine strain should firstly grow well and be safe in chicken embryos. However, most natural influenza virus strains are pathogenic to chicken embryos, and the growth titer of the virus is also low, which is difficult for mass production. Bird flu vaccine brings many inconveniences
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Embodiment 1
[0058] Embodiment 1, utilize reverse genetic manipulation technology of the present invention to carry out the rescue of influenza virus
[0059] 1. Construction of recombinant influenza virus in 8+7 mode
[0060] In this step, the 8+7 mode is used to rescue influenza virus, and the method is as follows: the cDNA molecule encoding PB2 in PR8, the cDNA molecule encoding PB1 in PR8, the cDNA molecule encoding PA in PR8, the cDNA molecule encoding NP in PR8, cDNA molecules encoding NA in PR8, cDNA molecules encoding M1 and M2 in PR8, cDNA molecules encoding NS1 and NS2 in PR8, and cDNA molecules encoding PB2 in YT, cDNA molecules encoding PB1 in YT, cDNA molecules encoding PA in YT 15 cDNA molecules, cDNA molecules encoding HA in YT, cDNA molecules encoding NP in YT, cDNA molecules encoding NA in YT, cDNA molecules encoding M1 and M2 in YT, and cDNA molecules encoding NS1 and NS2 in YT Each molecule is individually inserted into the eukaryotic cell expression vector to obtain 15...
Embodiment 2
[0143] Embodiment 2, vaccine seed virus immunogenicity and protective detection
[0144] 1. Detection of immunogenicity of vaccine seed virus
[0145] 10 SPF chickens were immunized with recombinant viruses numbered R1 and R2 with high fecundity in chicken embryos and inactivated vaccines of recombinant virus R37 obtained in 6+2 mode, and the remaining 10 chickens were injected with the same amount of PBS buffer liquid as a control.
[0146] 1. The preparation steps of inactivated vaccine are as follows:
[0147] 1) Add formaldehyde solution with a final concentration of 0.4% (volume percentage) to the recombinant virus solutions numbered R1, R2 and R37, and place them on a shaker at 37° C. for 48 hours to inactivate them.
[0148] 2) Take 96ml of the inactivated virus solution, add 4ml of Tween-80 dropwise as an emulsifier, and mix evenly by inversion.
[0149] 3) Preparation of oil phase: Take 200ml of white oil in a grinding bowl, add 2% aluminum stearate powder into it,...
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Abstract
The invention discloses a method for rescuing influenza virus. The method for rescuing the influenza virus comprises the following steps: 1) respectively inserting each of 15 cDNA molecules, namely cDNA molecules used for coding PB2, PB1, PA, NP, NA, M1, M2, NS1 and NS2 in an influenza virus chick embryo adapted strain and cDNA molecules used for coding PB2, PB1, PA, HA, NP, NA, M1, M2, NS1 and NS2 in a to-be-rescued influenza virus strain, into an eukaryotic expression vector, so that 15 recombinant expression vectors are obtained; and 2) introducing the 15 recombinant expression vectors into packaging cells, so that a recombinant influenza virus is obtained. The recombinant influenza virus vaccine strain obtained in the method for rescuing the recombinant influenza virus has high copying capability in chick embryo, growth titer in the chick embryo is also higher compared with the traditional vaccine, and antigenicity detection carried out on a vaccine seed virus finds that antigen property of the vaccine seed virus is not obviously different from a parent virus.
Description
technical field [0001] The invention relates to a method for rescuing influenza virus in the field of virus vaccine production, in particular to a preparation method for influenza virus vaccine strain with high fecundity in chicken embryos. Background technique [0002] H9N2 subtype avian influenza virus is a low-pathogenic avian influenza virus, which mainly infects chickens, ducks, geese, migratory birds and other poultry, but can cause a serious drop in egg production of laying hens, complex respiratory diseases in broilers and young chickens The H9N2 subtype avian influenza virus has become the main influenza virus subtype prevalent in chickens in my country. It is worth noting that the H9N2 subtype avian influenza virus can not only infect birds, but also can break through the interspecies barrier and infect mammals and humans without going through an intermediate host. Cases of human infection with H9N2 subtype avian influenza virus occur from time to time, highlightin...
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