肠道病毒71 VP1基因的原核表达及其抗血清的制备

The expression of EV71 VP1 gene and preparation of its antiserum

目的克隆并表达重组肠道病毒71(EV71)VP1基因,进行抗血清的制备并检测抗血清效价。方法利用原核表达系统将PCR获得的VP1片段构建成原核表达载体pET32a(+)-VP1,诱导其表达VP1融合蛋白并利用包涵体纯化方法进行重组蛋白的纯化,将纯化的重组蛋白免疫新西兰兔制备VP1抗血清,ELISA检测抗体效价。同时构建真核表达载体,利用其在真核细胞中的瞬时表达检测抗血清的特异性。结果通过克隆获得VP1原核表达载体,IPTG诱导VP1蛋白表达并纯化,SDS-PAGE结果显示重组蛋白表达且纯化浓度较高,将重组蛋白乳化后免疫新西兰兔3次,取血检测抗血清的效价,ELISA结果显示抗体效价为1∶64 000,利用所构建真核表达载体pcDNA3.1(+)-VP1表达VP1对抗血清进行检测,Western blot结果表明抗血清可较好的与VP1特异性结合。结论制备具有免疫原性的VP1蛋白及其效价较高的抗血清,为进一步研究抗EV71诊断方法、血清学诊断试剂盒及抗病毒疫苗的研制奠定基础。

EV71 is a main virus of hand-foot-moth disease, while VP1 has main antigenic determinant of EVT1. In this study, we aimed to prepare VP1 antiserum and detect its titer by cloning and expressing recombinant VP1 gene. Firstly, the prokaryotic expression vector pET32a （＋）-VP1 was constructed by using VP1 fragment from PCR product, and the recombinant protein expressed. Then the purified recombinant protein was used to immunize New Zealand rabbits for preparing VP1 antiserum. At the same time, a eukaryotic expression vector of VP1 was constructed with the aim of detecting the antiserum specificity. ELISA revealed that antibody titer of the prepared antiserum was 1：64 000; Western blot test of eukaryotic expression vector pcDNA3.1 （＋）-VP1 protein showed that the antiserum could specifically bind with VP1. Preparation of immunogenic VP1 protein and high titer antiserum has laid a foundation for further study on the methods of diagnosis and resistance vaccine.