文献类型：期刊文献

中文题名：吉富罗非鱼源无乳链球菌Sip-GAPDH融合基因的构建及其原核表达

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

作者：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]

机构：[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

年份：2014

卷号：30

期号：7

起止页码：137

中文期刊名：生物技术通报

外文期刊名：Biotechnology Bulletin

收录：CSTPCD、、北大核心2011、CSCD_E2013_2014、北大核心、CSCD

基金：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)

语种：中文

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

外文关键词：Streptococcus agalactiae;Sip-GAPDH fusion gene;SOEing PCR;Prokaryotic expression

中文摘要：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。

外文摘要：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.