牛冠状病毒S基因的序列分析及原核表达  

Sequence Analysis and Prokaryotic Expression of Bovine Coronavirus S Gene

作　　者：高国强[1] 王梦心 刘明明[1] 于仁冬 侯喜林[1] 周玉龙[1] 武瑞[1] 张国华[1] 刘琳珊[1] 任德强 GAO Guoqiang;WANG Mengxin;LIU Mingming;YU Rendong;HOU Xilin;ZHOU Yulong;WU Rui;ZHANG Guohua;LIU Linshan;REN Deqiang(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China;Harbin Vico Biotechnology Development Co.,Ltd.,Harbin 150000,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319 [2]哈尔滨维科生物技术开发有限公司,哈尔滨150000

出　　处：《中国畜牧兽医》2018年第7期1740-1749,共10页China Animal Husbandry & Veterinary Medicine

基　　金：兽医生物技术国家重点实验室开放课题(SKLVBF2018XX)

摘　　要：为了解牛冠状病毒(bovine coronavirus,BCoV)的S基因变异情况并建立ELISA检测方法,本研究对采自不同牛场的新生犊牛腹泻(CD)和成年牛冬痢(WD)腹泻样本提取总RNA,反转录合成cDNA,利用PCR扩增S全基因和S1基因。将S1基因目的片段连接表达载体pET-32a(+),并转化大肠杆菌BL21(DE3)感受态细胞,经PCR、双酶切及测序验证正确后,进行IPTG诱导表达。结果显示,CD与WD分离株S基因核苷酸为98.4%,CD和WD分离株与参考毒株BCoV-ENT株核苷酸同源性最高,分别为98.4%和98.5%,CD分离株与参考毒株SUN5株的同源性最低,为97.5%,WD分离株与FRA/EPI/Caen/2003/13同源性最低,为97.3%。通过比对可知,分离株与已知毒株之间存在较大差异,为疫苗候选毒株筛选提供依据。本试验同时构建了pET-32a-S1表达载体,在0.2mmol/L IPTG诱导5h时,重组菌能在大肠杆菌BL21感受态细胞中产生大量的S1融合蛋白,获得约58ku表达产物。本试验成功表达了S1蛋白,并对BCoV进行了核苷酸进化分析,为疫苗免疫效果评价方法的建立奠定了基础。In order to understand the variation of bovine coronavirus（BCoV）Sgene 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 Sand S1 genes were supplemented by PCR.The target fragment S1 was lighted into the expression vector pET-32 a（＋）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 Sgene of CD and WD isolates was 98.4%,and the isolates had the highest nucleotide homology with the reference strain BCoV-ENT,which were98.4% 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-32 a-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.

关 键 词：牛冠状病毒(BCoV) S基因 序列分析 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]