S7 gene and coded recombinant protein of grass carp reovirus strain, and acquisition method and application thereof
A reovirus, gene encoding technology, applied in the field of encoded recombinant protein and its acquisition, S7 gene, can solve the problems of transient immunity, easy to cause local or systemic reactions, limited source of raw materials, etc., and achieve good immune effect Effect
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Embodiment 1
[0024] Example 1 Extraction and RT-PCR amplification of grass carp reovirus strain GCRVGD108RNA
[0025] Extraction of viral RNA and synthesis of cDNA: 7 days after the grass carp fibroblasts were inoculated with the virus, the cells were repeatedly frozen and thawed 2 to 3 times, centrifuged at 4000 rpm for 30 min at 4 °C, the supernatant was taken, and the virus was precipitated by ultra-high speed centrifugation at 30,000 rpm. Trizol reagent was used to extract viral dsRNA, see the instructions of Trizol for the steps. The extracted dsRNA was detected by SDS-PAGE electrophoresis, and there were 11 clear bands. cDNA was synthesized by reverse transcription using "anchor primer" (Maan et al, 2007), and then amplified using S7-specific primers.
[0026] Add 3 μl cDNA single-stranded as template in every 20 μl PCR reaction system, PCR amplification, electrophoresis detection, find that the expected specific amplification band appears at about 1540bp place, recover this band ( ...
Embodiment 2
[0030] Example 2 Determination and Analysis of the S7 Gene Sequence of Grass Carp Reovirus Strain GCRVGD108
[0031] The recovered product was ligated with the pMD19-T Easy Vector vector, the ligated product was transformed into Escherichia coli (E.coli) DH5α, and positive clones were selected for sequencing. Blast homology analysis was performed on the sequence on NCBI, and the results showed that it had the highest homology with Muscovy duck cell adsorption protein σC and avian orthoreovirus σC, and the comprehensive score was as high as 43.5.
Embodiment 3
[0032] The construction of embodiment 3 recombinant S7 gene expression plasmids
[0033] A pair of primers were synthesized according to the sequences at both ends of the S7 gene, the upstream primer contained a BamH I restriction site, and the downstream primer contained an Xho I restriction site.
[0034] Upstream primer (F): 5'TCGC GGATCC TAATGGCCACTCGTGAC 3′ BamH I
[0035] Downstream primer (R): 5′TCTCCG CTCGAG CTTACAGCAAACTAC 3′Xho I
[0036] The pMD19-T plasmid containing the S7 gene was used as a template, and F and R were used as primers for PCR amplification, and a specific single band was obtained, and the product size was about 1540bp. The PCR amplified product was digested with BamH I / Xho I and connected to the prokaryotic expression vector pET30c. Transform E.coli DH5α, screen and pick positive clones, extract plasmids, and transform E.coli BL21(DE3). The recombinant plasmid was identified by double enzyme digestion with BamH I / Xho I, and the foreign gene ...
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