2型猪链球菌Rgg调控因子的序列结构分析及原核表达

Sequence and structure analysis of Rgg transcription regulator in Streptococcus suis serotype 2 and prokaryotic expression

目的克隆2型猪链球菌Rgg转录调控因子编码基因并进行原核表达和纯化,并对其进行生物信息学分析和结构预测。方法 PCR扩增2型猪链球菌中国强毒株05ZYH33基因组的rgg基因,构建重组表达质粒pQE30-rgg,转化大肠杆菌M15,筛选阳性转化子进行IPTG诱导表达,SDS-PAGE鉴定表达产物;确定最佳诱导条件后,大量培养诱导重组表达菌,Ni 2+亲和层析柱纯化重组蛋白,非变性PAGE电泳分析其体外聚合状态。结果整个Rgg蛋白由15个α螺旋和2个β转角组成;构建的重组质粒在宿主菌中可高效表达,15℃过夜诱导获得可溶性重组蛋白的比例最高;获得了较高纯度的Rgg重组蛋白,并证实其在体外可形成同源二聚体。结论成功地原核表达并纯化了Rgg重组蛋白,证明它存在二聚体结构,为进一步研究其调控机制奠定了基础。

To identify and demonstrated the properties of Rgg transcription regulator in highly virulent strains of Streptococcus suis serotype 2,the rgggene from the genomic DNA in the virulent strain 05ZYH33 was amplified by PCR and subcloned into plasmid pMD18-T and pQE30 with double digestion of BamHⅠand Hind Ⅲ.Subsequently,the prokaryotic expression plasmid pQE30-rggwas transformed to E.coli M15 after identification by restriction endonuclease digestion and DNA sequencing.Upon induction with IPTG,E.coli M15 containing the recombinant plasmid could express a distinct band with a molecular weight of 30 kDa,which was similar to the predicted band of Rgg protein as demonstrated by SDS-PAGE and Western blot.It was demonstrated that the recombinant protein expression could be the highest soluble yield after induction for overnight at 15 ℃.The recombinant Rgg protein was purified by Ni 2＋NTA affinity chromatography.Native-PAGE results showed that the purified protein forms homodimers in vitro,consistent with data for other members of the Rgg family.Secondary structure analysis displayed Rgg protein contained 15α-helices and 2β-turn.These results would be useful in the further studies on the function of Rgg transcription regulator.