Recombinant H7N9 subtype avian influenza virus-like particle, and preparation method and application thereof
A bird flu virus and particle technology, applied in the field of vaccines, can solve problems such as insufficient supply of chicken embryos, endogenous virus contamination, environmental pollution, etc.
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Embodiment 1
[0053] Embodiment 1: Construction of recombinant shuttle plasmid
[0054] The H7N9 subtype avian influenza virus A / Chicken / Guangdong / GD15 / 2016 (hereinafter referred to as GD15 strain) used in this experiment was preserved and provided by the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University. The strain has been published on the genebank sequence, the sequence number is: PB2 (KY751288), PB1 (KY751256), PA (KY751233), HA (KY751058), NP (KY751157), NA (KY751124), M (KY751091), NS (KY751190). According to the nucleic acid sequences of the HA, NA, and M1 genes of the avian influenza virus GD15 strain, the Primer Premier 5.0 software was used to design primers for gene amplification.
[0055] Table 1 Primer information for amplification of HA, NA and M1 genes
[0056]
[0057] Note: GCCACC (bold) represents the Kozak sequence; the underlined line is the restriction site.
[0058] The PCR reaction system is as follows...
Embodiment 2
[0066] Example 2: Rescue of recombinant baculovirus
[0067] The recombinant shuttle plasmids pVL-HA-GD15, pVL-NA-GD15, pVL-M1-GD15 and optimized baculovirus genomic DNA were co-transfected into Sf9 cells in logarithmic growth phase. The brief procedure is as follows:
[0068] 1) Add Sf9 cells to the six-well cell plate, 1×10 6 Place the cell plate horizontally in a 27°C incubator, incubate for 1 hour, and prepare transfection samples at the same time;
[0069] 2) Prepare the DNA transfection complex: Dilute the recombinant shuttle plasmid to a concentration of 100ng / μL for use; warm the transfection reagent to room temperature and mix gently; prepare two sterile EP tubes, add 100 μL serum-free respectively Culture medium; add 5μL (100ng) flashBAC to one tube TM For DNA, flick the EP tube to mix evenly; add 5 μL (500ng) transfer vector plasmid, flick the tube to mix evenly, then add 4 μL of transfection reagent, flick the EP tube to mix evenly, and centrifuge to precipitate;...
Embodiment 3
[0074] Example 3: Identification of recombinant baculovirus
[0075]1 PCR identification of exogenous genes in recombinant baculovirus genome
[0076] 1.1 Extraction of recombinant viral DNA
[0077] The extraction of recombinant baculovirus DNA was carried out according to the instructions of the whole gold virus extraction kit:
[0078] 1) Mix Poly A Carrier RNA with Binding Buffer 1:50, called MIX A;
[0079] 2) For each sample, take 200 μL supernatant, add 200 μL BB5 and 20 μL proteinase K, and vortex to mix;
[0080] 3) Incubate at 56°C for 15 minutes, add 250 μL of absolute ethanol, vortex and oscillate to mix, and then place at room temperature for 15 minutes;
[0081] 4) Install the adsorption column, transfer the mixed solution to the column, centrifuge at 12000rpm for 1min, and discard the lower liquid;
[0082] 5) Add 500 μL WB5, centrifuge at 12000 rpm for 1 min, discard the filtrate, and repeat this step once;
[0083] 6) Centrifuge at 12000rpm for 1min, disc...
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