摘要
目的:构建幽门螺杆菌vacA毒性片段(v)与hpaA融合基因的原核表达载体,并诱导表达,为制备具有治疗与预防作用的疫苗奠定基础. 方法:通过设计带有编码IL-3N端12个氨基酸引物,用PCR技术从pQE30-V质粒扩增出有接头的v基因,克隆至质粒pTrc99A-HpaA中与hpaA融合.将融合基因插入原核表达载体pQE30,再将pQE30-V-HpaA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65 000,能与幽门螺杆菌感染的人阳性血清发生抗原抗体反应. 结论:重组表达质粒pQE30-V-HpaA表达成功,为进一步研究其免疫学活性及制备疫苗提供了材料.
AIM: The prokaryotic expression vector of the fusion gene with v segment of the vacuolating cytotoxin and hpaA of Helicobacter pylori (H pylori) was constructed and expressed. It would lay a foundation for prophylaxis and therapy of H pylori infection. METHODS: By using the primer with a fragment encoding 12 amino acids of N-terminal of human interleukin-3 (IL-3), the vacuolating cytotoxin gene of Hp with linker was amplified from pQE30-V plasmid by PCR. The gene was cloned into plasmid pTrc99A-HpaA and fused with the hpaA gene. The fusion gene was cloned into prokaryotic expression vector pQE30. The recombinant plasmid of pQE30-V-HpaA was transformed into E.coli. DH5α and expressed in the presence of IPTG. The expression product was analyzed by SDS-PAGE, its antigenicity of the expression product was identified by Western blotting. RESULTS: Mr of recombinant protein was about 65 000 and represented 35% total protein of E.coli. Western blotting showed the recombinant protein could be recognized by the antiserum against H pylori. CONCLUSION: The fusion gene and its prokaryotic expression vector pQE30-V-HpaA is constructed and expressed in DH5α successfully. It provides the antigen basis for further studying the biological function of fusion protein and obtaining vaccine against the infection of H pylori.
出处
《世界华人消化杂志》
CAS
2004年第5期1096-1099,共4页
World Chinese Journal of Digestology
