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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 prokaryotic expression vector of vacuolar toxin VacA and adhesin HpaA in H.pylori cells and induce the expression of the fusion protein and to detect the antigenicity and immunogenicity of the expressed product. Methods VacA gene was amplified by PCR from pQE30-VacA plasmid, cloned into pTrc99A-HpaA vector, fused with HpaA gene, inserted into prokaryotic expression vector pQE30, and then transformed into E. coli DH5α, pQE30-VacA-HpaA was induced to express The fusion protein was purified, the fusion protein was detected by SDS-PAGE, the fusion protein content was detected by Bradford method, and the specificity was identified by Western blot. The fusion protein was immunized in rabbits to obtain polyclonal antiserum. Immunogenicity was detected by two-dimensional immunodiffusion and ELISA. Results SDS-PAGE showed that the relative molecular mass of the fusion protein was about 65000, the expression level was over 35%. The protein was mainly expressed in inclusion bodies with a protein content of 0.72 mg / ml. The fusion protein had good antigenicity and immunogenicity of VacA and HpaA. Conclusion VacA-HpaA fusion protein has been successfully expressed, and has good immunogenicity, which provides the conditions for further study of preparation of H. pylori vaccine.