肺炎链球菌表面黏附素A的原核表达和抗原性分析

Study on prokaryotic expression and antigenicity of pneumococcal surface adhesin A

目的应用基因工程技术制备肺炎链球菌表面黏附素A(pneumococcal surface adhesin A,PsaA),并分析其抗原性。方法根据基因库中报道的psaA基因序列,从肺炎链球菌基因组中扩增出psaA基因,连接到原核表达载体pET28a中,构建出重组质粒pET28a-psaA,转化大肠杆菌BL21(DE3),采用镍柱亲和层析法纯化PsaA蛋白,Western-blot分析其抗原性。结果克隆出的psaA基因同基因库中报道的psaA基因序列一致。成功构建出重组质粒pET28a-psaA,基因测序证实重组质粒构建正确。成功表达和纯化出PsaA蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示蛋白分子量为37-kDa,最终得到纯度较高的蛋白,主要以可溶性形式存在,Western-blot分析显示在37-kDa处见到目的蛋白条带。结论利用基因工程技术可以克隆出psaA基因,并成功表达和纯化出PsaA蛋白,其抗原性良好。

Objective To prepare the pneumococcal surface adhesin A（PsaA） with genetic engineering technology and to analyze its antigenicity. Methods Bank on the sequence ofpsaA gene in the Genebank,a pair of primers were designed and psaA gene was amplified from the genomes of Streptococcus pneumoniae. By gene recombination technology in vitro, sequence psaA was cloned into prokaryotic expression vector pET28a to construct pET28a- psaA recombinant plasmid. After being confirmed by DNA sequencing, expressed antigen protein was purified by affinity chromatography and the antigenicity of the expressed protein was confirmed by Western blotting assay. Results The cloned psaA gene was consistent with Genbank data by gone sequencing. A recombinant expression plasmid pET28a-psaA was constructed successfully and could stablely express about a 37-kD protein in E. coli BI21 （DE3） ,mainly exsited in the form of solution. The PsaA matigenicity was proved by Western blotting assay. Conclusions The psaA gene was successfully cloned, and the recombinant PsaA protein was expressed and purified, so that it can provide an experimental basis for the further research of the protein vaccine of otitis media.