sva-pcv2 fusion protein and its preparation method, gene, biological material, application and vaccine
A 1. SVA-PCV2, fusion protein technology, applied in the biological field, can solve the problems of infection, economic loss in aquaculture, and achieve the effect of strong antigenicity
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[0061] The present invention also provides a method for preparing the above-mentioned SVA-PCV2 fusion protein. The preparation method includes: expressing the gene encoding the SVA-PCV2 fusion protein in a host; system, insect expression system, plant expression system or mammalian expression system, preferably expressed in CHO-S cells.
[0062] In some preferred embodiments, the SVA-PCV2 fusion protein is prepared as follows:
[0063] (a) The VP1 polypeptide of SVA is connected to the PCV2Cap protein through flexible amino acids, and the gene encoding the fusion protein is artificially synthesized after optimization of partial tropism codons.
[0064] (b) The artificially synthesized gene was inserted into the eukaryotic expression vector pcDNATM3.1-TOPO, identified by restriction enzyme digestion and sequenced, and a positive recombinant vector was screened out to obtain the pcDNA3.1-SVA-PCV2-Cap plasmid.
[0065] (c) pcDNA3.1-SVA-PCV2-Cap plasmid using FreeStyle TM MAX R...
Embodiment 1
[0078] According to the SVA and PCV2 genomes obtained by logging into GenBank, the candidate gene sequences were finally determined through sequence comparison analysis. The polypeptide gene sequence of SVAVP1 was derived from GenBank: KY747519.1, and the gene sequence of PCV2Cap protein was derived from GenBank: FJ158606.
[0079] In this embodiment, two polypeptide genes of SVAVP1 are used, one is named SVA3, and the coding sequence of SVA3 is shown in SEQ ID NO.1; the other is named SVA5, and the coding sequence of SVA5 is shown in SEQ ID NO.2. Connect SVA3 and SVA5 to the PCV2Cap protein gene sequence end-to-end through the coding sequence of the connecting peptide, wherein the PCV2Cap protein gene sequence is shown in SEQ ID NO.3, and the coding sequence of the connecting peptide is shown in SEQ ID NO.4. After optimization of the tropism codon, restriction sites HindIII and BamHI were inserted at both ends of the gene, and sent to a synthetic company for gene synthesis to ...
Embodiment 2
[0081] To construct the pcDNA3.1-SVA-PCV2-Cap vector, the construction steps are as follows:
[0082] (a) Ligation: The recovered PCR product was ligated with pcDNA3.1. The ligation system (10 μL / tube) was as follows: 1 μL of pcDNA3.1 vector, 2 μL of recovered enzyme digestion product, dH 2 O 2 μL, Solution I 5 μL, and connect overnight on the connection instrument at 16°C.
[0083] (b) Preparation of Escherichia coli competent cells: a single colony was picked from a petri dish and inoculated into a test tube containing 10 mL of LB liquid medium, and cultivated 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 (200 μL) of pre-cooled CaCl 2 Buffer, gently suspend the c...
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