羊种布鲁氏菌Omp10基因的克隆及原核表达

›› 2014, Vol. 41 ›› Issue (12): 122-125.

羊种布鲁氏菌Omp10基因的克隆及原核表达

徐开莲, 朱华培, 赵天靖, 贾晓晓, 郭莳雨, 庞峰, 焦寒伟, 成鹰, 杜丽, 史巧芸, 荣辉, 张珈宁, 王凤阳

海南大学农学院, 海南省热带动物繁育与疫病研究重点实验室, 海口市动物基因工程重点实验室, 海南海口 570228

收稿日期:2014-04-28 出版日期:2014-12-20 发布日期:2014-12-25
通讯作者: 王凤阳 E-mail:fywang68@hotmail.com
作者简介:徐开莲(1989－), 女, 贵州人, 硕士生, 研究方向:动物功能基因组学.
基金资助:
"863"计划(2011AA100302、2013AA102524).

Cloning and Prokaryotic Expression of Omp10 Gene of Brucella melitensis

XU Kai-lian, ZHU Hua-pei, ZHAO Tian-jing, JIA Xiao-xiao, GUO Shi-yu, PANG Feng, JIAO Han-wei, CHENG Ying, DU Li, SHI Qiao-yun, RONG Hui, ZHANG Jia-ning, WANG Feng-yang

Key Laboratory of Animal Genetic Engineering of Haikou, Key Laboratory of the Tropical Animal Breeding and Epidemic Disease Research of Hainan, College of Agriculture, Hainan University, Haikou 570228, China

Received:2014-04-28 Online:2014-12-20 Published:2014-12-25

摘要/Abstract

摘要： 根据GenBank公布的羊布鲁氏菌(B.melitensis) M5-90株外膜蛋白(outer membrane protein,Omp)基因序列,设计1对引物,以其全基因组为模板,采用PCR技术对其进行扩增,得到381 bp的目的片段,连接入pMD20-T载体,转化E.coli DH5α感受态细胞;测序正确后,构建pET-28a-Omp10原核表达质粒,再将该质粒转化入E.coli BL21(DE3), IPTG诱导表达融合蛋白His-Omp10,用SDS-PAGE和Western blotting进行分析.结果表明, 成功构建了含Omp10基因的原核表达载体,并在E.coli BL21(DE3)中表达了Omp10基因,诱导得到的融合蛋白经鉴定与目的蛋白大小一致,证明Omp10得到成功表达.该试验为布鲁氏菌病的进一步研究奠定基础.

关键词: 布鲁氏菌; Omp10基因; 原核表达; 克隆

Abstract: According to the Brucella melitensis vaccine strain M5-90 outer membrane protein 10 (Omp10) genetic sequence in GenBank, a pair of primers was designed and the PCR technology was used to amplify Omp10 with its whole genome as the template. The Omp10 fragment which was 381 bp was obtained. Insert it into the plasmid pMD20-T, and it was transformed into E.coli DH5α. Recombinant prokaryotic expression plasmid, pET-28a-Omp10 was constructed, and transformed into E.coli BL21(DE3), and the fusion protein His-Omp10 was induced and expressed by IPTG, and identified with SDS-PAGE and Western blotting to analysis. The results showed that the recombinant protein was expressed successfully, which laid a solid foundation to the study of the animal immunization test profoundly.

Key words: Brucella melitensis; Omp10 gene; prokaryotic expression; cloning

中图分类号:

R378.5

徐开莲, 朱华培, 赵天靖, 贾晓晓, 郭莳雨, 庞峰, 焦寒伟, 成鹰, 杜丽, 史巧芸, 荣辉, 张珈宁, 王凤阳. 羊种布鲁氏菌Omp10基因的克隆及原核表达[J]. , 2014, 41(12): 122-125.

XU Kai-lian, ZHU Hua-pei, ZHAO Tian-jing, JIA Xiao-xiao, GUO Shi-yu, PANG Feng, JIAO Han-wei, CHENG Ying, DU Li, SHI Qiao-yun, RONG Hui, ZHANG Jia-ning, WANG Feng-yang. Cloning and Prokaryotic Expression of Omp10 Gene of Brucella melitensis[J]. , 2014, 41(12): 122-125.

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