牛冠状病毒S基因的序列分析及原核表达

doi:10.16431/j.cnki.1671-7236.2018.07.002

Abstract

Abstract:

In order to understand the variation of bovine coronavirus (BCoV) S gene and establish an ELISA detection method,total RNA was extracted from neonatal calf diarrhea (CD) and adult cattle winter dysentery (WD) diarrhea samples from different farms.cDNA was synthesized,and S and S1 genes were supplemented by PCR.The target fragment S1 was lighted into the expression vector pET-32a(+) and transformed into E.coli BL21 (DE3).The recombinant plasmid was induced by IPTG after being verified by PCR,double enzyme digestion and sequencing.The results showed that the nucleotide identitie of S gene of CD and WD isolates was 98.4%,and the isolates had the highest nucleotide homology with the reference strain BCoV-ENT,which were 98.4% and 98.5%,respectively.The homology of CD isolate with the reference strain SUN5 was the lowest,which was 97.5%,and the homology of WD isolate with strain FRA/EPI/Caen/2004/13 was the lowest,which was 97.3%.The comparison showed that there were a big difference between the isolated strains and the known strains,and provided a basis for screening the vaccine candidate strains.At the same time,pET-32a-S1 expression vector was constructed.The recombinant bacteria could produce a large amount of fusion S1 protein in E.coli BL21 and obtain 58 ku expression product,induced by 0.2 mmol/L IPTG for 5 h.In this study,S1 protein was successfully expressed,and nucleotide evolution analysis of BCoV were carried out,which laid the foundation for the establishment of the vaccine immune effect evaluation method.

Key words: bovine coronavirus (BCoV); S gene; sequence analysis; prokaryotic expression

CLC Number:

S852.65

GAO Guoqiang, WANG Mengxin, LIU Mingming, YU Rendong, HOU Xilin, ZHOU Yulong, WU Rui, ZHANG Guohua, LIU Linshan, REN Deqiang. Sequence Analysis and Prokaryotic Expression of Bovine Coronavirus S Gene[J]. , 2018, 45(7): 1740-1749.

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Sequence Analysis and Prokaryotic Expression of Bovine Coronavirus S Gene

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