|
|
|
口蹄疫病毒OH株结构蛋白基因VP1的克隆与表达
|
|
|
|
龚真莉1，2，刘湘涛1，尚佑军1，田宏1，吴锦艳1，尹双辉1，陈国栋1
|
|
|
|
（1.中国农业科学院 兰州兽医研究所 家畜疫病病原生物学国家重点实验室 农业部畜禽病毒学重点开放
实验室，甘肃 兰州730046; 2.甘肃农业大学，甘肃 兰州730070）
|
|
|
|
摘要：
|
|
采用RT-PCR方法扩增获得了O型口蹄疫病毒的主要免疫原VP1基因，将其插入pMD18-T载体进行序列分析，结果表明，所获得的基因片段含有完整的FMDV结构蛋白VP1编码区。根据表达载体pQE-Trisystem的克隆位点序列和该VP1基因片段的末端序列设计了1对表达引物，以重组pMD-T-VP1阳性质粒为模板，扩增获得了VP1基因，通过酶切将其克隆至表达载体pQE-Trisystem上。经测序证实，重组表达质粒所含的外源基因VP1编码框正确无误。将重组表达质粒pQE-VP1转化至大肠埃希氏菌M15，通过IPTG诱导促使VP1基因高效表达，SDS-PAGE和Western-blot分析表明，表达产物大小与预期的结果（26ku）一致，且具有良好的反应原性。以2mmol/L IPTG诱导表达5h时表达量最高，其中70%～80%的目的蛋白存在于菌体裂解后的上清中，表明外源基因VP1主要以可溶性方式表达。 |
|
关键词：
|
|
口蹄疫病毒；VP1基因；基因克隆；表达 |
|
中图分类号：S 852.659.6：Q 786 文献标识码：
A 文章编号：1673-4696(2006)07-0515-04 |
|
|
Cloning and expression of VP1 gene of foot-and-mouth disease virus |
|
|
|
GONG Zhen-li1，2,LIU Xiang-tao1,SHANG
You-jun1,TIAN Hong1,WU Jin-yan1,
YIN Shuang-hui1,CHEN Guo-dong1 |
|
|
|
(1.State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Animal Virology Ministry of Agriculture,
Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;
2.Gansu Agricultural University,Lanzhou 730070,China)
|
|
|
Abstract: The FMDV VP1 gene was amplified by RT-PCR, and then cloned into the vector pMD18-T. The sequencing result showed that the amplified fragment was the whole encoding region of the VP1 gene of FMDV. According to the characteristics of pQE-Trisystem and its restriction sites the appropriate expression primers were designed and the VP1 gene was ligated to the expression vector pQE-Trisystem. The sequence analysis showed that the VP1 gene was cloned to the expression vector successfully. The VP1 gene was expressed in E.coli M15 and induced by IPTG. SDS-PAGE and Western-blot analyses showed that the expressed VP1 protein had good antigenicity. When it was induced for 5 h with 2mmol/L of IPTG, the VP1 gene was expressed efficiently and 70%-80% of the protein was in suspension, indicating that the expressed VP1 protein was soluble. |
|
|
|
Key words: foot-and-mouth disease virus; VP1 gene; gene cloning; expression |
|
|
|
|
|
《中国兽医科学》 ©
版权所有
Chinese Veterinary Science
zgsykx@zgsykx.com |
