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| 牛冠状病毒S基因的序列分析及原核表达 | |
| 引用本文： | 高国强,王梦心,刘明明,于仁冬,侯喜林,周玉龙,武瑞,张国华,刘琳珊,任德强.牛冠状病毒S基因的序列分析及原核表达[J].中国畜牧兽医,2018,45(7):1740-1749. |
| 作者姓名： | 高国强 王梦心 刘明明 于仁冬 侯喜林 周玉龙 武瑞 张国华 刘琳珊 任德强 |
| 作者单位： | 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 |
| 基金项目： | 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0.2 mmol/L IPTG诱导5 h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58 ku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。 |
| 关 键 词： | 牛冠状病毒(BCoV) S基因 序列分析 原核表达 |
| 收稿时间： | 2017-11-14 |
Sequence Analysis and Prokaryotic Expression of Bovine Coronavirus S Gene |
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| 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].China Animal Husbandry & Veterinary Medicine,2018,45(7):1740-1749. | |
| Authors: | GAO Guoqiang WANG Mengxin LIU Mingming YU Rendong HOU Xilin ZHOU Yulong WU Rui ZHANG Guohua LIU Linshan REN Deqiang |
| Institution: | 1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China; 2. Harbin Vico Biotechnology Development Co., Ltd., Harbin 150000, China |
| 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. |
| Keywords: | bovine coronavirus (BCoV) S gene sequence analysis prokaryotic expression |
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