Influenza virus live vaccine and preparation method thereof
An influenza virus and live vaccine technology, applied in the field of virus vaccines, can solve the problems of insufficient cross-protection, risk of painful infection, weak cross-protection of influenza virus, etc. The effect of toxic phenotypes
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Embodiment 2
[0048] Example 2 Rescue of Recombinant Influenza Carrying rNA Gene
[0049] The rNA gene sequence designed in Example 1 was submitted to the biotechnology company, and the rNA gene double-stranded DNA was obtained by artificial synthesis. The synthetic gene was used as a template, primers were designed, and rNA amplification products containing SapI restriction sites at both ends were obtained by polymerase chain reaction. After the amplified product was digested with SapI, it was connected to the bidirectional expression vector pM to obtain the pM-rNA recombinant plasmid.
[0050] The constructed pM-rNA plasmid was co-transfected with the remaining 7 plasmids PM-PB2, PM-PB1, PM-PA, PM-HA, PM-NP, PM-M, and PM-NS of PR8 virus and co-cultured with 293H and MDCK cells. For specific experimental steps, refer to the instructions of Lipofectamine2000 (Invitrogen) transfection reagent. 48 hours after transfection, the transfection supernatant was collected and inoculated into 9-da...
Embodiment 3
[0051] Example 3 PR8-rNA virus biological characteristics analysis
[0052] 1) Identification of NAvRNA content in PR8-rNA virus genome
[0053] 1.1 Viral genome RNA electrophoresis detection: In order to detect the packaging efficiency of NA fragments, viral genome RNA electrophoresis was performed. Genomic RNA (vRNA) of purified virus was extracted using MagMAX Viral RNA Isolation Kit (Ambion). After the genomic RNA was extracted, the polyacrylamide (PAGE) gel with a concentration of 3.5% was used for RNA electrophoresis at 80V for 5 hours. After electrophoresis was completed, stained with SYBR Green II RNA Gel Stain (Invitrogen), and photographed. Compared with the wild-type PR8 virus (wtPR8), the concentration of NA vRNA bands in the recombinant virus PR8-rNA genome was significantly weakened (see image 3 ).
[0054] 1.2 Fluorescent quantitative PCR (Q-PCR) to measure the packaging efficiency of NA fragments:
[0055] Using the NP gene as an internal reference, the r...
Embodiment 4
[0073] Example 4 In vivo virulence detection of PR8-rNA virus
[0074] In order to further test the attenuated phenotype of PR8-rNA virus in vivo, we used the mouse infection model to evaluate the replication ability and pathogenicity of PR8-rNA influenza virus in mice. Female BALB / c mice aged 6-8 weeks (nine in each group) were inoculated with different doses of PR8-rNA and wtPR8 virus in nasal cavity, and the control group was added with PBS dropwise. On day 3 and day 6 after infection, 3 mice in each group were sacrificed, and the virus titers in nasal blades and lungs were determined. The body weight change and survival rate of the mice were observed for 14 consecutive days, and the weight change and survival curves of the mice were drawn.
[0075] 10 3 On the 2nd to 3rd day after the pfu wtPR8 virus infected the mice, the mice showed a typical onset state (depressed, disheveled, curled up, trembling), and their body weight decreased rapidly. All the mice died on the 5th...
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