以轮状病毒VP6为载体的RV/HBV嵌合蛋白的构建及抗原性研究(英文)

Construction of a rotavirus/hepatitis B virus plasmid and the immunogenic analysis of the expressed chimeric proteins

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 pETP6v carrier vector that expressed rotavirus（RV）VP6 protein and insert a gene fragment encoding the epitope of hepatitis B virus（HBV）S protein that expressed a RV/HBV chimeric protein,and to study the immunogenicity of this chimeric protein and assess its potential as a combined vaccine.Methods Using gene cloning and recombinant techniques,the gene of rotavirus VP6,the group（subgroup）of antigenic protein of the virus,was inserted into plasmid pETL to construct a carrier vector named pETP6v;then,a DNA fragment from HBV S gene that encodes the highly conserved epitope consisting of 21 amino acids（TTPAQGNSMFPSCCCSKPTDG）was inserted into the carrier vector pETP6v.The expression of this rotavirus VP6/HBV S epitope chimeric protein was analyzed in E.coli and further evaluated by ELISA and Western blot.Results A RV/HBV expression plasmid（pETP6/Hs）that carries the HBV S determinant was constructed and the RV/HBV chimeric protein expressed at very high levels in E.coli（about 36.2% of the total bacterial proteins）and the purity of the recombinant protein reached above 90%.The purified chemeric protein could react with anti-HBs serum from HBV-infected individuals and serum from guinea pigs immunized with rotavirus VP6 protein by ELISA and/or Western blot analysis.Conclusions The recombinant expression plasmid pETP6/Hs can be constructed to express chimeric proteins such as RV/HBV with high immunogenicity and reactivity and may be used in the development of RV/HBV and RV/other virus combined vaccines.