牛病毒性腹泻病毒GSTZ株E1基因原核表达载体的构建及表达  

Construction of Prokaryotic Recombinant Vector of E1 Gene of Bovine Viral Diarrhea and Its Induced Expression

作　　者：谢军 周昌娈 周小凯 马晓霞[1,2,3] 马忠仁 柏家林[1,2,3] XIE Jun;ZHOU Chang-luan;ZHOU Xiao-kai;MA Xiao-xia;MA Zhong-ren;BAI Jia-lin(ey Lab of Bioengineering and Biotechnology of the State Ethnic Affairs Commission, Northwest Minzu University,Lanzhou 730030 China;.Gansu Engineering Research Center,Northwest Minzu University,Lanzhou 730030 China;Fac Lty of Life Science and Engineering,Northwest Minzu University,Lanzhou 730030 China;Shanghai Veterinary Research Institute,Chinese Academy of Agric Ltural Science,Shanghai,200241 China)

机构地区：[1]西北民族大学生物工程与技术国家民委重点实验室,甘肃兰州730030 [2]西北民族大学甘肃省动物细胞工程技术研究中心,甘肃兰州730030 [3]西北民族大学生命科学与工程学院,甘肃兰州730030 [4]中国农业科学院上海兽医研究所,上海200241

出　　处：《西北民族大学学报（自然科学版）》2017年第4期46-51,共6页Journal of Northwest Minzu University(Natural Science)

基　　金：国家自然科学基金项目(31260533);中央专项研究生科研创新项目(Yxm2015207);甘肃省科技支撑计划项目(1604NKCA044);甘肃省兰州市科技计划项目(2016-3-120);兰州市城关区科技计划项目(2016-1-1)

摘　　要：为研究牛病毒性腹泻病毒的囊膜糖蛋白E1的抗原性,参考了Oregon C24V BVDV毒株的基因组序列(AF091605.1),并设计一对特异引物(上游引物5’-CCC GGA TCC ATG GCC TCT CCC TAC TGT-3’,下游引物5’-GGG CTC GAG TTA CCC TTG TGC TCC TGT T-3’),利用RT-PCR从病毒基因组中扩增E1基因609 bp全长片段,测序结果表明,与C24V株核苷酸同源性达100%.扩增产物经BamHI和XhoI双酶切,亚克隆至原核表达载体,双酶切鉴定表明成功构建了重组表达载体pET-32a(+)-E1.重组表达载体转化BL21(DE3)宿主菌,IPTG诱导表达,经SDSPAGE和Western-blot检测,成功诱导表达出25ku E1蛋白.实验结果为进一步研究BVDVE1蛋白的功能提供了参考.To study the antigenicity of E1 of cystic glycoprotein of the bovine viral diarrhea virus,A pair of special primers was designed for the genome sequence of the Oregon C24V BVDV strain(AF091605.1)(upstream primer 5'-CCC GGA TCC ATG GCC TCT CCC TAC TGT-3',downstream primer 5'-GGG CTC GAG TTA CCC TTG TGC TCC TGT T-3'),the total length of E1 gene was amplified by RT-PCR from the viral genome,the identification of sequencing showed that the nucleotide of C24V was 100%homologous.The amplified products were cut by BamH I and Xho I,subcloning to the prokaryotic expression vector,the dual-enzyme identification indicated that the recombinant expression vector pET-32a(+)-E1 was successfully constructed.Recombinant expression vector transformed BL21(DE3)host bacteria and IPTG induced expression were detected by SDS-PAGE and Western-blot.25 ku E1 proteins were successfully induced,and the results of the experiment provided references for further study of the function of BVDV E1 protein.

关 键 词：牛病毒性腹泻病毒 E1基因 原核表达 

分 类 号：S851[农业科学—预防兽医学]