Bovine viral diarrhea virus recombinant subunit vaccine
A technology for bovine viral diarrhea and viruses, which is applied in the field of human vaccines and human biological products, can solve the problems of large differences in virulence, low titer, and cattle infection, and achieve batch-to-batch stability and strong immunogenicity , the effect of high expression level
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[0038] An aspect of the embodiments of the present invention also provides a method for preparing a recombinant bovine viral diarrhea virus fusion protein, comprising:
[0039] Cloning of a eukaryotic expression vector comprising a recombinant bovine viral diarrhea virus fusion protein encoding gene;
[0040] Transfect CHO cells with the eukaryotic expression vector, and screen to obtain a CHO cell strain capable of stably and efficiently expressing the recombinant bovine viral diarrhea virus fusion protein in suspension, then ferment and culture the CHO cell strain, and then separate to obtain the recombinant Bovine viral diarrhea virus fusion protein;
[0041] The fusion protein has the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence more than 95% identical to the full-length amino acid sequence of SEQ ID NO:2.
[0042] In some embodiments, the eukaryotic expression vector includes but is not limited to pSV2-GS, pCI-GS or pcDNA4-GS, etc., preferably pCI-G...
Embodiment 1
[0065] Embodiment 1 Construction of recombinant eukaryotic expression vector pCI-Fu-GS
[0066] 1. Amplification and purification of BVDV-Fu gene The codon-optimized BVDV-Fu gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pUC-57 vector. For Kpn Ⅰ and Xho Ⅰ, the pUC-Fu plasmid vector was obtained. Use pUC-Fu as a template, and Fu-F and Fu-R as primers for PCR amplification (the gene sequences of Fu-F and Fu-R are shown in SEQ ID NO: 3 and SEQ ID NO: 4), and the amplification system See Table 1. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.
[0067] Table 1 BVDV-Fu gene amplification system
[0068]
[0069] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such as figure 1 As shown, a band...
Embodiment 2
[0080] Example 2 Construction and Screening of Recombinant CHO Cells Expressing BVDV-Fu Protein
[0081] 1. Cell Transfection
[0082] 1.1 Prepare cells Take CHO cells in the logarithmic growth phase, sample and count, and use 1×10 6 The cell density of cells / ml continued to be subcultured, the seeds were maintained, and the remaining cells were centrifuged at 1000 rpm for 4 minutes, then the supernatant was discarded, resuspended with about 20 ml of fresh CHO-WM medium, centrifuged again, and centrifuged at 1000 rpm for 4 minutes. Discard the supernatant and resuspend with a small amount of medium for counting, and finally adjust the cell density to 1.43×10 7 cells / ml.
[0083] 1.2 Plasmid and cell mixing Take 5 μg of the pCI-Fu-GS carrier obtained in Example 1, add it to the EP tube, add 0.7ml cells, mix well, and let stand for 15 minutes.
[0084] 1.3 Electroporation 280V 20 ms electric shock for 2 pulses. Immediately after the electric shock was completed, the cells wer...
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