文献类型：期刊文献

中文题名：罗非鱼源无乳链球菌ZQ0910转录调控因子rovS基因的克隆及表达研究

英文题名：Cloning and expression analysis of transcriptional regulator rovS gene of Stretococcus agalactiae ZQ0910 isolated from tilapia

作者：王蓓[1,2];简纪常[1,2];鲁义善[1,2];蔡双虎[1,2];黄郁葱[1,2];汤菊芬[1,2];李桂欢[1,2];吴灶和[2,3]

机构：[1]广东海洋大学水产学院,广东湛江524025;[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,广东湛江524025;[3]仲恺农业工程学院,广东广州510225

年份：2013

卷号：37

期号：4

起止页码：584

中文期刊名：水产学报

外文期刊名：Journal of Fisheries of China

收录：CSTPCD、、北大核心2011、Scopus、北大核心、CSCD、CSCD2013_2014

基金：国家重点基础研究发展计划(2009CB118704);国家重点基础研究发展计划前期(2011CB111600);广东省科技计划项目(2012B020308009);2011年广东省高等院校学科建设和教学质量与教学改革工程专项

语种：中文

中文关键词：无乳链球菌ZQ0910;RovS转录调控因子;克隆;原核表达

外文关键词：Stretococcus agalactiae ZQ0910; RovS transcription regulator; cloning; prokaryotic expression

中文摘要：为了对罗非鱼源无乳链球菌ZQ0910株毒力相关转录调控因子rovS进行克隆及表达研究,实验根据GenBank上登录的相关基因设计引物,采用PCR方法扩增该株细菌的rovS基因,然后将该基因定向克隆到原核表达载体pET-28a(+)中,在大肠杆菌Rosetta(DE3)中进行IPTG诱导表达。结果显示,该基因有849个碱基,编码282个氨基酸;同源基因序列比对显示,无乳链球菌ZQ0910株与无乳链球菌2 603 V与ATCC13813的rovS基因的同源性最高;经IPTG诱导后表达的融合蛋白分子量为34 ku;用亲和层析后的融合蛋白免疫新西兰大白兔制备多克隆抗体,经ELISA检测效价达到1∶512 000。研究结果表明,实验成功克隆与表达了rovS基因,为深入探讨RovS调节因子在调节细菌的代谢、生长和毒力等多种生命活动中的作用提供了理论依据。

外文摘要：Cloning and expression analysis of transcriptional regulator rovS gene of Stretococcus agalactiae ZQ0910 isolated from tilpia were studied in this study.A pair of primers were designed based on rovS gene sequence published in GenBank.The gene sequence of coding the RovS protein was cloned and then inserted into the pET-28（＋）vector to construct prokaryotic expression plasmid pET-28-RovS.The recombinant RovS fusion protein was overexpressed in E.coli Rosetta（DE3）cells in the presence of isopropyl-β-thiogalactopyranoside（IPTG）.Sequence analysis revealed that rovS gene is 849 bp and encodes a putative protein of 282 amino acids,and the amino acid sequence of RovS of S.agalactiae ZQ0910 showed highest identity of S.agalactiae 2 603 V and ATCC13813.The His-RovS fusion protein with 34 ku molecular mass was successfully expressed in E.coli Rosetta（DE3）.The soluble recombinant protein was highly expressed under induction conditions of exposure to IPTG and successfully purified on Ni2＋-IDA column.The purified fusion protein was injected into New Zealand white rabbits to produce polyclonal anti-RovS serum.The antibody titer detected by ELISA reached 1∶ 512 000.In summary,we identified and characterized a novel regulatory gene,termed rovS,encoding a regulator of virulence in S.agalactiae,which is involved in the expression of known and putative virulence genes in the bacteria.