白念珠菌磷酸甘油酸激酶1的原核表达及免疫原性分析

Prokaryotic expression and immunogenicity analysis of phosphoglycerate kinase 1 of Candida albicans

目的构建白念珠菌基因磷酸甘油酸激酶1(PGK-1)基因的原核表达质粒并诱导其表达,对其免疫原性进行分析。方法 PCR法获取白念珠菌SC5314的PGK-1基因,插入原核表达载体pET30a中,以重组质粒转染感受态细胞E.coli BL21,经IPTG诱导表达后,SDS-PAGE检测蛋白的表达情况,并用Western blot法验证。用纯化获取的重组蛋白免疫BALB/c小鼠,ELISA对蛋白的免疫原性进行评价。结果成功构建原核表达质粒pET-30a-pgk-1,诱导后表达的重组蛋白相对分子质量(M r)约为54 810。ELISA检测抗体效价约为1∶1 024。结论成功获得白念珠菌PGK-1重组蛋白,该蛋白具有良好的免疫原性。

Objective To construct prokaryotic expression plasmids of Candida albicans gene phosphoglycerate kinase 1 （pgk-1） and examine the immunogenicity of the recombinant protein. Methods The full-length coding sequence of pgk-1 was amplified by PCR and cloned into the prekaryotic expression vector pET30a. The 6 x His-tagged protein was induced by IPTG in E. coli BL-21 （DF_.3） and the recombinant protein was identified by SDS-PAGE and Western blotting. The BALB/c mice were immunized with the purified recombinant protein to evaluate the antigenicity of the recombinant protein by ELISA. Results The full-length pgk-1 gene was cloned from SC5314 genome and pET-3Oa-pgk-1 was successfully constructed. The recombinant protein PGK-1 was highly expressed in E. coli with a relative molecule mass of 54 810. ELISA indicated that the titer of the antibody was about 1：1024. Conclusion PGK-1 was successfully expressed by prokaryotic expression system and the recombinant protein showed favorable immunogenicity in mice.