马麝出血症病毒VP60主要抗原表位区的串联表达及对家兔的免疫保护效力分析

Tandem expression of the major epitope domains of the Moschus chrysogaster hemorrhagic disease virus VP60 and its protective efficacy to rabbits

马麝病毒性出血症(Moschus chrysogaster viral hemorrhagic disease,McVHD)为马麝的一种急性、高度致死性传染病,其病原马麝出血症病毒(Moschus chrysogaster hemorrhagic disease virus,McHDV)与兔出血症病毒高度同源。为了筛选McHDV保护性抗原,为McVHD疫苗研究奠定基础,文中通过对McHDV主要结构蛋白VP60抗原表位的分析,设计引物,应用RT-PCR技术扩增获得三段VP60主要抗原表位区核酸序列,并采用重叠延伸PCR将3段产物连接后克隆至原核表达载体pET-28a(+),成功构建原核表达质粒pET-truncated-VP60后转化大肠杆菌Escherichia coli BL21(DE3),并经IPTG诱导表达。纯化表达产物免疫3–4月龄非RHD免疫兔,血凝抑制试验测定抗血清效价。首次免疫后21 d,通过攻毒保护试验分析重组蛋白的免疫保护效力。结果表明,McHDV VP60主要抗原表位蛋白在大肠杆菌中成功表达,重组蛋白分子质量约为45 kDa,且以包涵体形式存在;重组蛋白纯化后配制疫苗免疫的新西兰白兔,可100%抵抗McHDV株的攻击,表明所筛选的抗原表位串联表达的重组蛋白具有良好的免疫保护效力,研究结果为McVHD新型疫苗的研发奠定了基础。

Moschus chrysogaster(sifanicus)viral hemorrhagic disease(McVHD)is an acute and highly lethal infectious disease caused by Moschus chrysogaster hemorrhagic disease virus(McHDV)whose genome sequence is highly homologous with rabbit hemorrhagic disease virus.To screen the protective antigen of McHDV and set the basis for study of McVHD vaccine,the antigen epitope of major structural protein VP60 of McHDV was analyzed,and the specific primers were designed to obtain three amplified DNA sequences encoding the main antigen epitope of VP60 from McHDV by using RT-PCR.Then the three DNA fragments were sequenced and cloned to prokaryotic expression vector with pET-28 a(+)by using overlap extension PCR,and finally the prokaryotic expression plasmid pET-truncated-VP60 was constructed.Subsequently,the pET-truncated-VP60 was transformed into Escherichia coli BL21(DE3),and the recombinant proteins were expressed by IPTG induction.Finally,the expressed protein was purified and applied to immunize that without immunizing with RHD vaccine,then the antiserum titers were evaluated by the hemagglutination inhibition test,and the immune-protective efficacy of the recombinant proteins was observed and analyzed through animal challenge test.The results showed that the multi-epitope DNA fragments of VP60 of McHDV was successfully expressed in the form of inclusion bodies in E.coli,and the relative molecular weight of recombinant proteins is about 45 kDa.After immunized with the recombinant proteins,100% of New Zealand white rabbits were resistant to attack of McHDV,which indicates efficient immune-protective efficacy of chosen epitope recombinant protein.The study laid a foundation for the development of the new subunit vaccines of McVHD.