兔瘟病毒VP60蛋白原核表达及其应用

Prokaryotic Expression of VP60 Gene of Rabbit Hemorrhagic Disease Virus and Its Application

为获得活性良好的兔出血症病毒基因工程抗原,本研究对兔出血症病毒ZB分离株的VP60基因进行了原核表达与初步应用。参照ZB株分离病毒VP60基因序列,设计合成一对特异性引物,PCR扩增长876bp的VP60基因片段。将目的片段定向克隆至pET30a表达载体中,经鉴定正确后,重组质粒转化BL21表达菌,经IPTG诱导后获得了以包涵体形式表达的重组蛋白,重组蛋白纯化后,Western blot检测表明具有良好的抗原性与特异性,以该蛋白作为诊断抗原,初步建立了检测兔瘟病毒抗体的间接ELISA诊断方法。本研究为RHDV分子流行病学调查提供了参考,为VP60蛋白结构与功能研究、RHDV抗体检测试剂盒及新型疫苗的研制奠定基础。

In order to obtain good activity of gene engineering, the research were prokaryotic expres- sion of rabbit hemorrhagic disease virus ZB strain VP60 gene. According to ZB strains of VP60 gene se- quence, one pairs of primers were designed, 876 bp fragment of VP60 gene were amplified by PCR. The fragment was directionally cloning into pET30a expression vector, after identified correct, the recombinant plasmid was transformed into BL21 expression bacterium and induced by IPTG. to get the recombinant protein expressed in inclusion body forms. After the recombinant protein was purified, Western blot detec- tion showed that the expressed protein had good antigenicity and specificity. Using this protein as diagnos- tic antigen, an indirect ELISA detection method was established preliminary for rabbit haemorrhagic dis- ease virus antibody detection. The research provides a reference for RHDV molecular epidemiology study, which Lays a foundation for the research of structure and function of VP60 protein, RHDV antibody detec- tion kit research and development of new vaccines.