HA-Fc fusion protein and preparation method and vaccine thereof
A fusion protein, ha-fc technology, applied in the field of biomedicine, can solve the problems of low immunogenicity, low yield, high pathogenicity, etc., and achieve the effect of high immunogenicity
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Embodiment 1
[0077] Avian influenza HA-Fc gene synthesis:
[0078] The avian influenza HA gene was obtained according to the login to GenBank, and the gene sequence was obtained from GenBank: ABW90137.1. After optimization of codon preference, the HA gene sequence is shown in SEQ ID NO.1. The sequence of the Fc region of porcine IgM is shown in SEQ ID NO.2, and the sequence of the Fc region of human IgG is shown in SEQ ID NO.3. The HA gene was spliced with the Fc region of porcine IgM and the Fc region of human IgG respectively; a His tag with 6 histidines was added to the N-terminus, restriction sites HindIII and BamHI were inserted at both ends of the gene, and sent to the synthetic company Perform gene synthesis.
Embodiment 2
[0080] pcDNA TM 3.1-HA-Fc plasmid construction
[0081] 1. Ligation reaction: Ligation of the recovered PCR product and pcDNA3.1, the ligation system (10 μL / tube) is as follows: 1 μL of pcDNA3.1 vector, 2 μL of the recovered enzyme digestion product, ddH 2 O 2 μL, Solution I 5 μL, and connect overnight on the connection instrument at 16°C.
[0082] 2. Preparation of Escherichia coli competent cells: Pick a single colony from a petri dish and inoculate it into a test tube containing 10 mL of LB liquid medium, and culture overnight at 37°C. Take 100 μL of the bacterial solution and transfer it to a centrifuge tube containing 10 mL of liquid LB medium, incubate with shaking at 37°C for 2 hours; divide the bacterial solution into 1.5mL centrifuge tubes, and place in an ice bath for 20 minutes; centrifuge at 4,000 rpm for 5 minutes at 4°C, discard the supernatant ;Add 1 / 5 volume (200μL) of pre-cooled CaCl 2 Buffer, gently suspend the cells; centrifuge at 4000rpm at 4°C for 5min,...
Embodiment 3
[0088] Preparation of HA-Fc fusion protein by CHO expression system
[0089] 1. Cell Culture
[0090] CHO-S cells, cultured at 37°C, 5% CO 2 , 125rpm, and the culture medium (Hycell+8mM GlutaMAX) was purchased from HyClone Company. FreeStyle TM MAX reagent was purchased from Invitrogen Company. The density and viability of CHO-S cells were counted and observed by trypan blue staining. CHO-S cell suspension culture, cell density 1.5×10 6 / mL, transfection was performed when the cell viability was ≥90%. Cultured in a shaking shaker incubator, culture parameters: 37 ° C, 5% CO 2 , 125rpm.
[0091] 2. Plasmid extraction: through PureLink TM The instructions of Hipure Plasmid Maxiprep Kit (endotoxin-free) kit require extraction of recombinant plasmids for transfection.
[0092] 3. Transfection: pcDNA TM 3.1-HA-Fc plasmid using FreeStyle TM MAX Reagent was transfected into CHO-S cells, and the cell supernatant was collected after 14 days to obtain the desired antigen. ...
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