鸡传染性支气管炎病毒S1蛋白的真核表达及免疫原性分析

Eukaryotic Expression and Immunogenicity Analysis of Avian Infectious Bronchitis Virus S1 Protein

为真核表达鸡传染性支气管炎病毒(IBV)的S1蛋白并鉴定其免疫原性,构建了IBV广西优势血清型代表株GX-YL5 S1蛋白基因的重组表达载体pFastBacTM/HBM-TOPO-S1,转化DH10Bac细胞进而拯救出重组杆状病毒;经间接免疫荧光(IFA)和Western blot鉴定并大量表达;纯化后免疫新西兰大白兔制备兔抗血清,应用IFA和Western blot对该重组蛋白的反应原性进行鉴定,并应用气管环(TOC)中和试验分析其免疫原性。IFA显示重组杆状病毒感染后72 h细胞出现特异性荧光;Western blot显示重组蛋白与抗His标签单克隆抗体以及IBV特异性抗体均能结合,且反应性良好,特异性强;中和试验显示制备的血清对IBV的中和滴度为1∶512。表明该重组S1蛋白具有较好的免疫原性,为IBV S1蛋白的生物学功能、基因工程亚单位疫苗等研究奠定了基础。

In order to express the S1 protein of avian infectious bronchitis virus(IBV)in eukaryotic expression and identify its immunogenicity,the recombinant expression vector pFastBacTM/HBM-TOPO-S1 containing the S1 protein gene of IBV strain GX-YL5,the Guangxi representative dominant serotype isolate of IBV was constructed and transfected into DH10Bac cells,and then the recombinant baculovirus was rescued from the Sf9 cells infected with rHBM-S1.The expression products were identified by indirect immunofluorescence(IFA)and Western blot.After identification,a large amount of recombinant S1 protein was expressed in suspension culture Sf9 cells.The recombinant S1 protein was purified and used to immunize New Zealand white rabbits to prepare anti-rabbit serum.IFA and Western-blot were used to identify the reactivity of the recombinant protein.The immunogenicity was analyzed by trachea organ ring(TOC)neutralization test.The result of IFA showed that the specific fluorescence of recombinant baculovirus cells occurred 72 h after infection.Western blot analysis showed that the fusion recombinant protein combined with anti-His-labeled monoclonal antibody and IBV-specific antibody.The rabbit polyclonal antibody prepared by recombinant S1 protein reacted with the expressed recombinant S1 protein with good reactivity and specificity.The neutralization test result indicated that the neutralization titer of the prepared rabbit antiserum was 1∶512.In summary,the results showed that S1 protein of IBV GX-YL5 strain was expressed successfully in eukaryotic expression system,and the recombinant S1 protein had good immunogenicity.This study laid a foundation for the studies on the biological function of IBV S1 protein and genetic engineering subunit vaccine.