羊种布鲁氏菌Wzt基因的克隆及原核表达

›› 2014, Vol. 41 ›› Issue (11): 54-57.

羊种布鲁氏菌Wzt基因的克隆及原核表达

庞峰, 贾晓晓, 赵天靖, 朱华培, 徐开莲, 郭莳雨, 史巧芸, 荣辉, 周海龙, 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:2014-06-03 出版日期:2014-11-20 发布日期:2014-12-06
通讯作者: 王凤阳 E-mail:fywang68@163.com
作者简介:庞峰(1987-),男,山东人,硕士生, 研究方向:动物功能基因组。
基金资助:
"863"计划 (2011AA100302、2013AA102524)。

Cloning and Prokaryotic Expression of Wzt Gene of Brucella melitensis

PANG Feng, JIA Xiao-xiao, ZHAO Tian-jing, ZHU Hua-pei, XU Kai-lian, GUO Shi-yu, SHI Qiao-yun, RONG Hui, ZHOU Hai-long, WANG Feng-yang

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

Received:2014-06-03 Online:2014-11-20 Published:2014-12-06

摘要/Abstract

摘要： 为进一步研究Wzt蛋白在光滑型脂多糖合成路径中的作用,本试验利用PCR技术,以羊种布鲁氏菌16 M株基因组为模板,扩增出大小为759 bp的Wzt基因片段,将其连入pMD20-T载体,测序正确后构建重组质粒pET-28a-Wzt,转化E.coli BL21(DE3)工程菌,IPTG诱导其表达,最后用Western blotting鉴定蛋白。结果显示,扩增出的Wzt基因片段大小为759 bp,与GenBank中登录的羊种布鲁氏菌16 M株Wzt基因序列(登录号:AF047478.1)同源性为99.87%,证明成功克隆了Wzt基因,同时成功构建了pET-28a-Wzt原核表达载体,并在E.coli BL21(DE3)工程菌中表达了Wzt蛋白,诱导得到的融合蛋白大小约为30 ku,位于25～35 ku之间,与目的蛋白大小一致,结果表明成功表达了目的基因。

关键词: 羊种布鲁氏菌; Wzt基因; 克隆; 原核表达

Abstract: In order to further study the role of Wzt protein in the synthesis of smooth lipopolysaccharide, the PCR technology was used to amplify the Wzt gene of 759 bp .Then it was ligated into pMD20-T vector.The recombinant plasmid pET-28a-Wzt was transformed into E.coli BL21(DE3) for expression under induction of IPTG. The protein product was analyzed by SDS-PAGE and Western blotting. The results showed that the size of the Wzt gene was 759 bp and the sequence homology was 99.87% compared with the sequence of Brucella melitensis 16 M strain with the access number of AF047478.1 in GenBank,proving that the Wzt gene was successfully cloned. Also, the prokarotic expression vector pET-28a-Wzt was successfully constructed and the Wzt protein with molecular weight of 30 ku was highly expressed in E.coli BL21(DE3), which all the above proved that Wzt gene was successfully expressed.

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

中图分类号:

庞峰, 贾晓晓, 赵天靖, 朱华培, 徐开莲, 郭莳雨, 史巧芸, 荣辉, 周海龙, 王凤阳. 羊种布鲁氏菌Wzt基因的克隆及原核表达[J]. , 2014, 41(11): 54-57.

PANG Feng, JIA Xiao-xiao, ZHAO Tian-jing, ZHU Hua-pei, XU Kai-lian, GUO Shi-yu, SHI Qiao-yun, RONG Hui, ZHOU Hai-long, WANG Feng-yang. Cloning and Prokaryotic Expression of Wzt Gene of Brucella melitensis[J]. , 2014, 41(11): 54-57.

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