Ⅱ型B族链球菌表面免疫相关蛋白基因的克隆和原核表达

Cloning and Prokaryotic Expression of Gene Encoding Surface Immunogenic Protein(SIP) of Streptococcus Group B

目的重组表达Ⅱ型B族链球菌表面免疫相关蛋白(Surfaceimmunogenicprotein,SIP)基因,为进一步免疫学研究提供目标蛋白。方法用PCR的方法从GBSⅡ型标准株的基因组DNA中扩增出SIP基因,用T/A克隆法将其插入pMD18T载体,构建原核表达载体pET32aSIP,用BL21(DE3)/pET系统表达TrixSIP融合蛋白,SDSPAGE和质谱分析鉴定表达产物,并对表达蛋白进行初步纯化。结果PCR扩增产物经测序,证实与GenBank中Ⅰa/c型GBS的SIP的基因序列同源性为99%。SDSPAGE显示,经IPTG诱导后BL21(DE3)/pET32aSIP总蛋白中出现一条相对分子质量为66000的新蛋白带。质谱分析和蛋白质库的比较证实其为B族链球菌表面免疫相关蛋白(SIP)的可能性分数为74。结论已成功表达并初步纯化SIP,为SIP在细菌致病中的作用研究以及相关疫苗的制备奠定了基础。

Objective To express the gene encoding the surface immunogenic protein（SIP） of streptococcus group B and provide target protein for further immunological study. Methods Amplify SIP gene from the genomic DNA of standard GBS strain type Ⅱ by PCR and insert into pMD18-T vector by T/A cloning. Digest the recombinant plasmid with restriction endonuclease Bgl Ⅱ and Hind Ⅲ, and clone the obtained gene fragments into pET32a vector. Transform the constructed recombinant plasmid pET32a-SIP into BI21 （DE3）/pET system for expression of Trix-SIP fusion protein. The expressed product was identified by SDS-PAGE and peptide mass fingerprinting（PMF）. Results The homology of amplified SIP gene sequence to that of SIP of GBS type Ia reported in GenBank was 99%. SDS-PAGE showed a protein band with relative molecular weight of 66 000. The probability based mowse score of SIP was 74 by PMF analysis. Conclusion GBS SIP fusion protein was successful expressed. It laid a foundation of study on role of SIP in pathopoiesis of bacteria and development of relevant vaccine.