吉富罗非鱼源无乳链球菌GAPDH融合基因的构建及其原核表达

生物技术通报 ›› 2014, Vol. 0 ›› Issue (7): 137-142.

吉富罗非鱼源无乳链球菌GAPDH融合基因的构建及其原核表达

王蓓^{1, 2, 3}, 李桂欢^{1, 2, 3}, 鲁义善^{1, 2, 3}, 汤菊芬^{1, 2, 3}, 黄郁葱^{1, 2, 3}, 吴灶和^{2, 3, 4}, 简纪常^{1, 2, 3,}

1.广东海洋大学水产学院, 湛江 524088；
2.广东省水产经济动物病原生物学及流行病学重点实验室, 湛江 524088；
3.广东省水产经济动物病害控制重点实验室, 湛江 524088；
4.仲恺农业工程学院, 广州 510225

收稿日期:2013-12-16 出版日期:2014-07-15 发布日期:2014-07-16
作者简介:王蓓, 女, 博士, 助理研究员, 研究方向：水产经济动物病原生物学；E-mail：wong19820204@126.com
基金资助:
国家自然科学基金青年基金项目（31302226）, 广东省科技计划农业攻关项目（2012B020308009）, 广东省2012年鱼病防治专项（2130108）

Construction and Prokaryotic Expression Sip-GAPDH Fusion Gene of Streptococcus agalactiae from GIFT Strain of Nile Tilapia（Oreochromis niloticus）

Wang Bei ^{1, 2, 3}, Li Guihuan^{1, 2, 3}, Lu Yishan ^{1, 2, 3}, Tang Jufen ^{1, 2, 3}, Huang Yucong ^{1, 2, 3}, Wu Zaohe ^{2, 3, 4}, Jian Jichang ^{1, 2, 3}

1. Fisheries College, Guangdong Ocean University, Zhanjiang 524088；
2. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088；
3. Guangdong Key Laboratory of Control for Diseases of Aquatic Economic Animals, Zhanjiang 524088；
4. Zhongkai University of Agriculture and Engineering, Guangzhou 510225

Received:2013-12-16 Published:2014-07-15 Online:2014-07-16

摘要/Abstract

摘要： 利用重叠延伸（SOEing）PCR技术, 将吉富罗非鱼（Oreochromis niloticus）源无乳链球菌（Streptococcus agalactiae）ZQ0910株表面免疫原性蛋白（Surface immunogenic protein, Sip）基因与甘油醛-3-磷酸脱氢酶（GAPDH）基因通过Linker序列融合, 构建成为Sip-GAPDH融合基因。将其定向克隆至原核表达载体pET-32a（+）中, 在大肠杆菌BL21（DE3）中进行诱导表达。结果表明, 重组质粒pET-32a-Sip-GAPDH在大肠杆菌BL21（DE3）中表达后所获得的融合蛋白Sip-GAPDH分子量为102.01 kD, 表达最佳条件为37℃, IPTG浓度0.1 mmol/L, 诱导5 h。Western blot鉴定结果表明融合基因得到了成功表达。

关键词: 无乳链球菌, Sip-GAPDH融合基因, 重叠延伸PCR技术, 原核表达

Abstract: The DNA fragment encoding the Sip and GAPDH gene of Streptococcus agalactiae strain ZQ0910 isolated from GIFT Strain of Nile Tilapia（Oreochromis niloticus）were obtained by PCR. The Sip-GAPDH gene was constructed in a Sip-linker-GAPDH format with the standard 15-amino acid linker（Gly4Ser）3 by SOEing PCR technique, and the final full length product was cloned into the pET-32a（+）vector for protein expression in Escherichia coli strain BL21（DE3）. The result showed that the expression fusion protein Sip-GAPHD were about 102.01 kD and Western blotting analysis confirmed that the 102.01 kD protein was the fusion protein, because it was specifically recognized by mouse anti-His monoclonal antibody. Inducing the cells at 37℃ in 0.1 mmol/L of IPTG for 5 hours was the optimal conditions for expression of the recombinant Sip-GAPDH fusion protein.

Key words: Streptococcus agalactiae, Sip-GAPDH fusion gene, SOEing PCR, Prokaryotic expression

王蓓, 李桂欢, 鲁义善, 汤菊芬, 黄郁葱, 吴灶和, 简纪常. 吉富罗非鱼源无乳链球菌GAPDH融合基因的构建及其原核表达[J]. 生物技术通报, 2014, 0(7): 137-142.

Wang Bei , Li Guihuan, Lu Yishan , Tang Jufen , Huang Yucong , Wu Zaohe , Jian Jichang. Construction and Prokaryotic Expression Sip-GAPDH Fusion Gene of Streptococcus agalactiae from GIFT Strain of Nile Tilapia（Oreochromis niloticus）[J]. Biotechnology Bulletin, 2014, 0(7): 137-142.

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