肺炎链球菌表面黏附素A的原核表达及免疫保护性研究

Prokaryotic expression and protective effects of Pneumococcal surface adhesin A of streptococcus Pneumoniae

目的利用基因工程技术获取原核表达的肺炎链球菌表面黏附素A(PsaA)蛋白并初步研究其对小鼠的免疫保护性。方法根据基因库中psaA基因序列设计合成特异性引物,从临床分离到的肺炎链球菌中提取DNA,PCR技术扩增目的基因psaA,克隆后构建原核表达载体PET-32a(+)/psaA,将重组质粒转化到大肠杆菌BL21(DE3)中,以IPTG诱导表达,获得重组蛋白,并通过Western-blot鉴定其免疫活性。将重组蛋白免疫小鼠,观察对肺炎链球菌感染动物的保护作用。结果经酶切及测序鉴定,克隆出870bp的目的基因,DNA序列与Gen-Bank中的相应数据符合率为100%;成功构建出原核表达载体pET-32a/psaA;经IPTG诱导,表达出了约57kD左右的融合蛋白,与预期大小相符,并且具有抗体结合活性;重组PsaA蛋白对肺炎链球菌感染小鼠具有一定保护作用。结论在原核细胞中成功表达出PsaA蛋白,能在一定程度上抵抗肺炎链球菌感染,为基于黏附素的新型疫苗的研究提供线索。

[ Objectives ] To obtain PsaA by prokaryotic expression system and investigate its protective effect against infection of Streptococcus pneumoniae in mice. [Methods ] The specific primers were designed and produced according to PsaA of Streptococcus pneumoniae gene sequence. Template DNA was isolated and psaA gene was cloned by PCR. PCR product was cloned into PGM-T vector. The recombinanting plasmid PGM-T/psaA was cured with two kind of enzyme, the fragement was ligated to the express vector pET32a （＋） to construct recombinanting express vector pET32a （＋）/psaA. Eventually, recombinating plasmid were expressed after transformation in Escherichia coli BL21 （DE3）. Recombinating fusion protein was detected with SDS-PAGE and Western-blot. After actively immunized mice were challenged with Streptococcus pneumoniae, we observed middle survive times. [ Results ] psaA DNA secquence consistented with the data of Gene-Bank. Expressed fusion protein was confirmed PsaA and displayed satisfactory antigenicity. PsaA stimulated immunity to infection by Streptococcus Pneumoniae. [ Conclusions ] Protein PsaA is successfully expressed, it can product immunity to infection by Streptococcus Pneumoniae . PsaA may be a neotype candidate vaccine about adhesion.