猪链球菌2型38000蛋白主要功能区基因的克隆与表达

Cloning and expression of main regions of 38 000 gene of Streptococcus suis serotype 2 in Escherichia coli

根据GenBank中已发表的猪链球菌2型38000蛋白基因核苷酸序列,设计合成1对特异性引物,采用PCR方法,以四川分离株猪链球菌2型基因组DNA为模板扩增38000蛋白主要功能区基因。将PCR产物纯化后与pMD18-T连接转化宿主菌DH5α,提取阳性质粒,进行PCR和酶切鉴定。将目的片段定向克隆到表达载体pET32a中,经测序正确后,重组质粒转化入大肠杆菌BL21(DE3),于37℃、0.8mmol/LIPTG条件下进行诱导表达,结果重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子质量约为35000的重组蛋白,表达产物经纯化后,免疫印迹法(Western blotting)证实该重组蛋白可以与猪链球菌2型阳性血清发生特异性反应。

According to the published 38 000 protein gene sequence of Streptococcus suis （SS2）,a pair of primers was designed and synthesized. The main regions of the 38 000 gene was amplified from geneomic DNA of Streptococcus suis serotype 2 strain from Sichuan province by PCR and was cloned into pMD18-T vector. The plasmid of positive clones was extracted,digested with enzymes and detected by PCR. The PCR products were later cloned into plasmid vector pET32a via restrication endonuclease and then transformed into E. coli BL21 （DE3）, which were expressed respectively by 0.8 mmol/L IPTG of optimal concentration at 37 ℃ overnight. The fusion protein with a molecular weight of about 35 000,which the recombinant protein can react with positive serum against SS2. These results could be expressed using SDS-PAGE electrophoresis. Western blotting showed that provide the foundation for the practical application of the recombinant protein.