传染性法氏囊病病毒主要结构蛋白VP2、VP1和VP2-VP1串联基因的原核表达及其初步应用

为获得传染性法氏囊病病毒(IBDV)特异性抗体检测用抗原VP2、VP1及VP2-VP1蛋白,分别设计引物扩增IBDV野毒株NN1172的VP2和VP1基因,并扩增VP2和VP1基因中抗原性和亲水性较好的重要区域,通过PCR扩增基因串联方法对截短的VP2和截短的VP1基因进行串联,首次获得VP2-VP1串联基因,并对VP2、VP1和VP2-VP1串联基因进行了原核表达和鉴定。结果成功构建了原核表达载体pET-VP2、pET-VP1和pET-VP2-VP1;诱导表达条件显示,3个重组质粒分别转入BL21菌株后经0.05 mmol/L IPTG诱导表达,分别得到分子量为69、114和63 kDa的VP2、VP1和VP2-VP1重组蛋白,且均以包涵体形式表达,3个重组蛋白分别于诱导后5、3和6 h时表达量最多。Western blot结果显示,表达的VP2、VP1和VP2-VP1蛋白与鸡抗IBDV阳性血清均具有良好的反应原性。以纯化的VP2、VP1和VP2-VP1蛋白作为包被抗原对传染性支气管炎病毒(IBV)、呼肠孤病毒(ReoV)、禽白血病病毒(ALV)和新城疫病毒(NDV)4种阳性血清检测均为阴性,表明所获得的纯化蛋白具有高度的特异性;对免疫了IBD灭活疫苗,IBD基因工程疫苗和IBD弱毒疫苗的商业鸡群进行抗体检测,结果均能显示疫苗免疫后机体抗体水平的变化趋势。本研究表明利用该原核表达系统所表达的3个蛋白均具有良好的免疫反应活性,为IBDV特异性抗体的检测和新型亚单位疫苗的研发奠定基础。

Prokaryotic expression of VP2,VP1 and VP2-VP1 multimer genes of infectious bursal disease virus and its preliminary clinical application

In order to obtain VP2,VP1 and VP2-VP1 antigens against chicken infectious bursal disease virus(IBDV)for the antibody detection,primers were designed to amplify the VP2 and VP1 genes of an IBDV field strain NN1172,and the important segments with antigenicity and hydrophilicity in the VP2 and VP1 genes were predicted and screened.The gene multimer of VP2-VP1 was obtained for the first time by PCR amplification.And prokaryotic expression and identification of the VP2,VP1 and VP2-VP1 multimer genes were performed.The prokaryotic expression vectors pET-VP2,pET-VP1 and pET-VP2-VP1 were successfully constructed.The conditions of induced expression showed that the three recombinant plasmids were transfected into the E.coli BL21 strain and induced by 0.05 mmol/L IPTG,and the recombinant proteins of VP2,VP1 and VP2-VP1 with molecular weights of 69 k Da,114 k Da and 63 k Da were obtained,respectively.The expression levels of the three recombinant proteins were the highest at 5 h,3 h and 6 h after induction,respectively,and all of them were expressed as inclusion bodies.Western blotting analysis showed that the expressed proteins VP2,VP1 and VP2-VP1 had good antigenicity with chicken anti-IBDV positive serum.Four positive sera of IBV,Reo V,ALV and NDV were detected using the purified VP2,VP1 and VP2-VP1 proteins as coating antigens,and the results were all negative,indicating that the purified proteins were highly specific.An antibody test was carried out on commercial chickens immunized with the IBD inactivated vaccine,the IBD genetically engineered vaccine and the IBD attenuated vaccine;and the results showed the trend of changes of the antibody levels after vaccine immunization.In addition,184 serum samples detected in parallel with commercial kits A and B showed that the VP2 protein,as a coating antigen,had the highest relative sensitivity and coincidence rate.This study demonstrated that the VP2,VP1 and VP2-VP1 proteins expressed by the prokaryotic expression system had good immunoreactivity and might be used for IBDV antibody detection,which provided good antigens for the specific detection of antibody and the development of new subunit vaccines.