Recombinant baculovirus expressing manually modified and synthesized influenza A H1N1 virus HA-NA-M1 gene
A technology of influenza virus and type A, applied in the field of virology, can solve the problems of ineffective prevention of new influenza viruses and increased disease risk, and achieve the effects of easy monitoring, increased expression level, and improved screening efficiency
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Embodiment 1
[0022] Example 1: Construction of a recombinant baculovirus QP-Ac-HNM1 expressing the artificially modified and synthesized influenza A H1N1 virus HA-NA-M1 gene.
[0023] 1. Synthesis of main immunogenic genes of H1N1 influenza virus.
[0024] Referring to the HA, NA and M1 gene sequences of the H1N1 influenza virus A / California / 04 / 2009 (H1N1) strain, the sequences of the HA, NA and M1 genes were artificially synthesized according to the codon preference, wherein the NA and M genes were synthesized Together, the promoter (PH) of the baculovirus polyhedron gene is added at the N end of the NA gene, the poly(A) sequence of the SV40 virus is introduced at its C end, and the promoter PH is added at the N end of the M1 gene.
[0025] 2. Construction of the baculovirus transfer vector pFBDPHmHNM1P10eGFP.
[0026] According to the backbone of the baculovirus vector pFastBacDual in the Bac-to-Bac Baculovirus Expression System (kit) of Invitrogen, the polyA of chicken globin is insert...
Embodiment 2
[0034] Example 2: Identification of recombinant baculovirus QP-Ac-HNM1, preparation of VLP vaccine and immunogenicity experiment.
[0035] 1. Identification of recombinant baculovirus QP-Ac-HNM1.
[0036] The sf9 cells were inoculated into 6-well cell culture plates, and when the cells grew to 80-90% monolayer, they were inoculated with 1 MOI of recombinant baculovirus QP-Ac-HNM1, and a control baculovirus was set at the same time, and observed daily. After 72 hours of infection, more than 90% of sf9 cells glowed green fluorescence.
[0037] Take 400 μl of culture supernatant and cell suspension, treat with trypsin at 56°C for 4 hours, extract twice with an equal volume of phenol / chloroform, take the supernatant and add 1 / 10 volume of 3M sodium acetate and 2 times volume of Precipitate with absolute ethanol, centrifuge to discard the supernatant, wash the precipitate once with 500 μl 70% ethanol, dry it naturally, add 50 μl sterilized deionized water to resuspend and use it a...
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