幽门螺杆菌VacA-HpaA融合蛋白的表达及其免疫学特性

Expression and Immunological Characteristic of VacA-HpaA Fusion Protein of Helicobacter pylori

目的构建H.pylori细胞空泡毒素VacA与黏附素HpaA融合基因的原核表达载体,诱导其表达融合蛋白,并检测表达产物的抗原性与免疫原性。方法用PCR从pQE30-VacA质粒扩增出VacA基因,克隆至pTrc99A-HpaA载体中,与HpaA基因融合后,插入原核表达载体pQE30中,再将pQE30-VacA-HpaA转化入大肠杆菌DH5α,诱导表达并提纯融合蛋白,SDS-PAGE检测融合蛋白的表达,Bradford法检测融合蛋白含量,Western blot鉴定特异性。将融合蛋白免疫家兔,得到多克隆抗血清,用双向免疫扩散和ELISA检测免疫原性。结果SDS-PAGE显示融合蛋白相对分子质量约为65000,表达量在35%以上,主要以包涵体形式表达,蛋白含量为0·72mg/ml,具有良好的VacA和HpaA抗原性与免疫原性。结论VacA-HpaA融合蛋白已成功表达,且具有良好的免疫原性,为进一步研究制备H.pylori疫苗创造了条件。

Objective To construct the prokaryotic expression vector for fusion gene of VacA and HpaA of Helicobacter pylori, induce the expression of VacA-HpaA fusion protein and study the antigenicity and immunogenicity of expressed product. Methods Amplify VacA gene from pQE30-VacA plasmid and fuse with the HpaA gerre in vector pTrc99A-HpaA. Insert the fusion gene into prokaryotic expression vector pQE30,then transform the constructed recombinant plasmid pQE30-VacA-HpaA to E. coli DH5α for expression of VacA-HpaA fusion protein. Purify the expressed product by Ni^2＋ -NTA affinity chromatography and identify by SDS-PAGE. Analyze the content of fusion protein by Bradford method and the specificity by Western blot. Immunize rabbits with the fusion protein to prepare polyclonal antiserum and determine for immunogenicity by double immunodiffusion test and ELISA. Results SDS-PAGE showed that the fusion protein with a relative molecular weight of about 65 000 was expressed in a form of inclusion body and contained more than 30% of total somatic protein. The expressed product ,at a protein content of 0.72 mg/ml ,showed good antigenicity and immu- nogenicity of both VacA and HpaA. Conclusion VacA-HpaA fusion protein was successfully expressed. It laid a foundation of further development of H. pylori vaccine.