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目的构建轮状病毒(rotavirus,RV)VP6载体及携带乙型肝炎病毒(hepatitis B virus,HBV)外源抗原表位的嵌合质粒,进一步探索研发携带HBV抗原表位的重组嵌合疫苗的技术。方法运用基因克隆和重组技术,构建轮状病毒VP6载体,同时将HBV编码中和抗原表位21个氨基酸的基因序列构建到VP6载体上相应酶切位点,在大肠杆菌中表达嵌合蛋白,并进行相关免疫学性质的研究。结果重组嵌合蛋白rVP6/Hs可在大肠杆菌系统中高效表达,表达的蛋白占菌体总蛋白的36.2%,纯度90%以上;ELISA结果显示,嵌合蛋白可与抗-HBs抗体反应;Western blot实验表明,嵌合蛋白可分别与抗VP6豚鼠血清抗体和乙型肝炎患者血清抗体特异性结合。结果表明所表达的嵌合蛋白具有较好的抗原反应性。结论成功构建载体质粒pETP6v及携带HBV外源抗原表位的嵌合质粒pETP6/Hs,对研究病毒抗原表位具有重要的价值;所构建和表达的携带HBV S抗原表位的重组嵌合蛋白在研发RV/HBV重组嵌合蛋白疫苗方面具有重要潜在应用前景。
Objective To construct a rotavirus (VP6) vector and a chimeric plasmid carrying exogenous epitopes of hepatitis B virus (HBV), and to further explore the development of a recombinant chimeric vaccine carrying HBV epitopes . Methods The gene of rotavirus VP6 was constructed by gene cloning and recombination technology. At the same time, a 21 amino acid sequence encoding HBV neutralizing epitope was constructed on the corresponding restriction site of VP6 vector, and the chimeric protein was expressed in E. coli. And related immunological properties of the study. Results The recombinant chimeric protein rVP6 / Hs was highly expressed in E.coli. The expressed protein accounted for 36.2% of the total bacterial proteins and the purity was over 90%. ELISA showed that the chimeric protein could react with anti-HBs antibody. Western The result of blot showed that the chimeric protein could specifically bind to the anti-VP6 guinea pig serum antibody and the hepatitis B antibody respectively. The results showed that the expressed chimeric protein has good antigen reactivity. Conclusion The recombinant plasmid pETP6v and the chimeric plasmid pETP6 / Hs harboring HBV foreign epitopes have been successfully constructed and have important value in the study of viral epitopes. The recombinant chimeric protein carrying HBV S antigen epitope R & D RV / HBV recombinant chimeric protein vaccine has important potential applications.