布鲁氏菌外膜蛋白16基因的克隆及原核表达

›› 2013, Vol. 40 ›› Issue (9): 51-54.

布鲁氏菌外膜蛋白16基因的克隆及原核表达

贾晓晓, 焦寒伟, 郭莳雨, 史巧芸, 荣辉, 张珈宁, 朱华培, 杜丽, 成鹰, 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:2012-12-11 出版日期:2013-09-20 发布日期:2013-09-18
通讯作者: 王凤阳
作者简介:贾晓晓(1990-),女,河南人,硕士生,研究方向:动物功能基因组学。
基金资助:
"十二五"农村领域国家科技计划课题(2011AA100302);"863"项目(2013AA102524)。

Cloning and Prokaryotic Expression of Outer Membrane Proteins 16 Gene of Brucella melitensis

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

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

Received:2012-12-11 Online:2013-09-20 Published:2013-09-18

摘要/Abstract

摘要： 为了成功克隆外膜蛋白16(outer membrane proteins 16, Omp16)基因并对其进行原核表达,试验根据GenBank中羊布鲁氏菌M5-90株外膜蛋白Omp16基因序列(登录号:JF918760.1)设计1对引物,从布鲁氏菌基因组中扩增出大小约为507 bp的目的基因片段,凝胶回收纯化目的片段,连接入pMD20-T质粒,转化E.coli DH5α并测序,测序正确后再亚克隆入pET-28a(+)表达载体,构建重组质粒pET-Omp16,转化入E.coli BL21(DE3),经IPTG诱导其表达,最后用Western blotting分析方法鉴定诱导得到的蛋白。结果表明,成功构建了pET-Omp16原核表达载体,并在E.coli BL21中表达了Omp16基因,诱导得到的蛋白经鉴定与目的蛋白大小一致,证明成功表达了目的基因。

关键词: 布鲁氏菌; 外膜蛋白16基因; 克隆; 原核表达

Abstract: To successfully clone the outer membrane proteins 16 (Omp16) gene and make prokaryotic expression in E.coli, one pair of primers was designed according to B. melitensis M5-90 strain Omp16 gene sequence in GenBank, and then obtained Omp16 gene which was about 507 bp by PCR from the Brucella genome. After purifying, Omp16 gene was inserted into pMD20-T vector to construct recombinant plasmid pMD-Omp16. pMD-Omp16 transformed into E.coli DH5α and identified it by sequencing, then subcloned to vector pET-28a(+). The constructed recombinant plasmid pET-Omp16 was transformed into E.coli BL21(DE3) for expression under induction of IPTG. Lastly, the expression products of recombinant protein His-Omp16 was identified by Western blotting. The results showed that the prokaryotic expression vector was successfully constructed and expressed Omp16 gene in E.coli BL21.

Key words: B.melitensis; Omp16 gene; cloning; prokaryotic expression

中图分类号:

贾晓晓, 焦寒伟, 郭莳雨, 史巧芸, 荣辉, 张珈宁, 朱华培, 杜丽, 成鹰, 王凤阳. 布鲁氏菌外膜蛋白16基因的克隆及原核表达[J]. , 2013, 40(9): 51-54.

JIA Xiao-xiao, JIAO Han-wei, GUO Shi-yu, SHI Qiao-yun, RONG Hui, ZHANG Jia-ning, ZHU Hua-pei, DU Li, CHENG Ying, WANG Feng-yang. Cloning and Prokaryotic Expression of Outer Membrane Proteins 16 Gene of Brucella melitensis[J]. China Animal Husbandry & Veterinary Medicine, 2013, 40(9): 51-54.

参考文献

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