摘要
目的构建表达幽门螺杆菌融合蛋白粘附素-细胞空泡毒素A-霍乱毒素B亚单位(HpaA-CtxB-VacA,HCTV)的原核表达载体,并诱导表达,为制备具有治疗与预防作用的多联疫苗奠定基础。方法用PCR技术从pQE-hctB扩增hpaA和ctxB目的基因片段,从pQE30-V质粒扩增出vacA基因,同时插入表达载体pQE-30,构建成pQE-hctv。再将pQE-hctv转化大肠杆菌DH5α,经IPTG诱导表达。SDS-PAGE分析表达结果。Western blotting鉴定其抗原性。结果融合蛋白的相对分子量约为68000,可溶性表达占全菌的15%以上,经亲和层析后可获得纯度为90%以上的重组蛋白。Western blotting分析其能分别与HpaA抗血清、VacA抗血清和CT抗血清特异性反应。结论重组表达质粒pQE30-hctv表达成功,而且具有各自蛋白的抗原性,为进一步研究融和蛋白的功能和制备集预防和治疗为一体的Hp候选口服疫苗提供基础。
The prokaryotic expression vector for gene encoding the fusion protein HCTV [adhesion A of H. pylori （hpaA）, B-segment of the vacuolating cytotoxin of H. pylori （VacA）, A-subunit of cholera enterotoxin （Ctx-B）] was constructed and ex- pressed to lay a foundation for prophylaxis and treatment of H. pylori infection. For this purpose, the target genes for hpaA and CtxB were amplified from plasmid pQE-hcB with PCR technique, and VacA gene was amplified from plasmid pQE-30-V. These genes were inserted into prokaryotic expression vector pQE-30 to construct pQE-hctv, and then transformed into E. coli DHSα cells. The gene products were expressed after IPTG induction, as the fusion protein HCTV and analyzed by SDS-PAGE. Its immunogencity was identified by Western blotting. It was found that the relative molecular weight of this fusion protein was approximately about 68000 with the purity of more than 90 96 as demonstrated by affinity chromatography. This fusion protein could be specifically recognized by anti-HpaA, anti-VacA and anti CT antisera. It is concluded that the recombinant expression plasmid pQE-30-hctv was successfully constructed and expressed with definite immunogenicity and specificity, thus providing the basis for the candidate antigen to be used in the oral vaccine against H. pylori infection.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2006年第2期97-100,共4页
Chinese Journal of Zoonoses
基金
云南省教育厅科技基金资助课题(批准号03Y620C)
