Asia 1型口蹄疫病毒VP1基因的表达鉴定及其多抗制备

Expression of VP1 gene of foot-and-mouth disease virus serotype Asia 1 and antiserum preparation

本研究旨在表达口蹄疫病毒(FMDV)的VP1全基因并制备特异性的多克隆抗体。利用PCR方法扩增Asia 1 IND 49197株VP1全基因,将其克隆至原核表达载体pET-30a(+)中,在大肠杆菌BL21中进行表达。SDS-PAGE结果显示表达产物分子量约为31.6ku,以包涵体的形式存在。通过Ni-NTA Purification System纯化后进行western blot和间接ELISA分析,结果显示重组蛋白能够被FMD阳性血清识别,具有良好的反应性。将纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,ELISA测定抗体效价为1∶20480,病毒中和试验测定抗体效价为1∶64。本研究所表达的VP1蛋白可用于开发检测Asia1口蹄疫抗体的诊断试剂,所制备的多克隆抗体为进一步研究VP1的结构、功能以及抗原表位的鉴定提供了条件。

In this study,the VP1 gene of foot and mouth disease virus（FMDV） was amplified from Asia 1 IND 4/2004 strain by PCR and cloned into the pET-30a（＋） for expression in E. coli BL21. The expressed protein had a molecular weight of approximately 31.6 ku,and could react specifically to FMD positive serum in indirect ELISA and western blot analysis. Polyclonal antibodies to the VP1 protein was prepared by inoculating rabbits with purified recombinant VP1 protein followed by boosting 3 times. Anti-FMDV-VP1 serum were generated with antibody titres of 1∶20 480 in ELISA and 1∶64 in virus neutralization test. The recombinant protein obtained in this study could be used for detection of FMDV Asia 1 antibodies,and the polyclonal antibodies could be used to further study the structure,function and epitope mapping of FMDV serotype Asia 1 VP1 gene.