幽门螺杆菌UreB-NAP-HpaA三价DNA疫苗的构建及免疫原性初步研究

目的:构建幽门螺杆菌(Hp)尿素酶亚单位B(UreB)、中性粒细胞激活蛋白(NAP)及黏附素A(HpaA)三价重组DNA疫苗并研究其抗原性,为Hp疫苗的开发奠定基础.方法:PCR扩增UreB、NAP、HpaA全长序列,分别克隆入pMD 18-T载体,测序并鉴定方向后依次连接,构建融合基因UNH,然后将其亚克隆入真核表达载体pIRES,以此转染COS-7细胞,Western印迹检测表达蛋白的抗原性.结果:PCR分别获得约1 707、432和750 bp产物,与GenBank中相关序列具有高度同源性,三者融合后获得一约2 901 bp目的基因.重组质粒pIRES-UNH转染COS-7细胞后表达相对分子质量约107 000的蛋白质,Western印迹显示该重组蛋白可为UreB、NAP、HpaA抗血清特异识别,具有良好的抗原性.结论:成功构建了具有良好免疫原性的UreB-NAP-HpaA三价重组DNA疫苗,为进一步探讨其免疫保护性及Hp疫苗的研制奠定了基础.

Construction and immunogenicity study of a trivalent DNA vaccine candidate with UreB-NAP-HpaA of Helicobacter pylori

Objective:To construct a recombinant trivalent DNA vaccine of Helicobacter pylori (H. pylori) urease subunit B (UreB), neutrophil-activating protein (NAP) and adhesin A (HpaA), and study its antigenicity so as to lay a foundation for developing an H. pylori vaccine. Methods: Complete UreB, NAP and HpaA gene sequences obtained by PCR amplification from H. pylori genome DNA were cloned into pMD 18-T vector, and the products were identified and fused to abtain UNH. UNH was then subcloned into an eukaryotic expression vector pIRES. The identified recombinant plasmid pIRES-UNH was transfected into COS-7 cells for target protein expression. Immunogenicity of the recombinant fusion protein was analysed by Western blot. Results: PCR produced fragments about 1 707 bp, 432 bp and 750 bp, which had high homology with those related sequences in GenBank. With PCR and restriction enzyme digestion, a recombinant eukaryotic expression plasmid pIRES-UNH containing the fusion gene of the 3 fragments ( about 2 901 bp) was constructed. A new protein strip, with a relative molecular weight about 107 000, was produced by COS-7 cells transfected with pIRES-UNH. Western blot showed that the protein had a specific reaction with UreB, NAP and HpaA antiserum. Conclusion: A UreB-NAP-HpaA trivalent recombinant DNA vaccine with good immunogenicity has been successfully constructed, which lays a foundation for further investigation on its immunoprotective activity.