鸡传染性支气管炎病毒GX-YL5株S蛋白在昆虫杆状病毒表达系统的表达及其免疫原性

Expression of the S protein of avian infectious bronchitis virus strain GX-YL5 in insect baculovirus expression system and its immunogenicity analysis

为了对鸡传染性支气管炎病毒(avian infectious bronchitis virus,IBV)广西优势血清型代表株GX-YL5的S蛋白进行真核表达并研究其免疫原性,设计GX-YL5毒株S基因特异引物,扩增出目的片段后,构建重组表达载体pFastBacTM/HBM-TOPO-S,转化DH10Bac细胞获得重组杆状病毒rHBM-S;重组S蛋白鉴定正确后,大量表达、纯化并免疫新西兰大白兔制备兔抗血清,应用IFA、Western blot以及间接ELISA、气管环(TOC)中和试验对该重组蛋白的反应原性及免疫原性进行分析。结果显示,成功获得重组S蛋白;制备的抗S蛋白多抗血清具有良好的特异性,ELISA效价可达1∶25600,TOC中和试验滴度为1∶512。结果表明,应用昆虫杆状病毒表达系统成功表达了具有很好免疫原性的GX-YL5株的S蛋白。本试验为研究IBV S蛋白的生物学功能、研发诊断试剂和制备新型疫苗等奠定了基础,为利用昆虫杆状病毒表达系统进行IBV或其他冠状病毒结构蛋白的表达及多抗的制备提供了借鉴和参考。

In order to express the S protein of Guangxi representative dominant serotype isolate of avian infectious bronchitis virus(IBV)strain GX-YL5 in eukaryotic expression system and identify its immunogenicity,S gene specific primers of IBV strain GX-YL5 were designed and S gene was amplified.The recombinant expression vector pFastBacTM/HBM-TOPO-S was constructed and transformed into DH10 Bac cells to obtain the recombinant bacmid rHBM-S,and then the recombinant baculovirus was rescued from the Sf9 cells infected with rHBM-S.After being identified as correct,the recombinant S protein was expressed in large quantities and purified,and used to immunize New Zealand white rabbits to prepare rabbit antiserum.The reactivity and immunogenicity of the recombinant S protein were analyzed by indirect immunofluorescence assay(IFA),Western blot,ELISA and trachea organ ring(TOC)neutralization test.The results showed that the recombinant S protein was successfully expressed,the prepared rabbit antiserum has good reactivity and specificity,and the antibody titer of antiserum against the S protein was up to 1∶25600 by ELISA.The neutralization titer was 1∶512 by TOC neutralization test.The results indicated that the recombinant S protein of IBV strain GX-YL5 was successfully expressed in the insect baculovirusexpression system and had good immunogenicity.This study not only lay agood foundation for study the biological function of IBV S protein,the development of diagnostic reagents and new vaccine,but also provides a reference for the expression of IBV or other coronavirus structural proteins and the preparation of polyclonal antibodies using insect baculovirus expression system.