摘要
根据已发表的鸭甲肝病毒(Duck hepatitis A virus,DHAV)疫苗株A66株基因组序列,利用生物学分析软件DNAStar进行VP1蛋白的二级结构和抗原域预测,确定其主要抗原域(major antigen domain of VP1,VP1M),设计并合成一对可扩增VP1M的特异性引物,PCR扩增获得长为327 bp的VP1M基因片段。将VP1M基因片段定向克隆至p GEX-4T-1表达载体中,鉴定正确后进行IPTG诱导表达,获得了以包涵体为主的重组蛋白。重组蛋白纯化后,经过Western blot检测表明重组蛋白具有良好的抗原性。以该蛋白作为包被抗原,成功建立了检测DHAV-1的间接ELISA方法。该方法具有良好的特异性、敏感性和重复性,为DHAV的快速诊断、流行病学调查和免疫鸭群的抗体检测提供了快速实用的检测手段。
The published complete genome sequence of VP1 of Duck hepatitis A virus （DHAV） strain A66 was used to predict its secondary structure and antigen domain and to determine its major antigen domain of VP 1 （VP 1 M） using molecular biological software DNAStar. Accordingly, a pair of specific primers were designed and synthesized, which were used for amplification of the VP1M gene fragment with 327 bp in length. The VP1M gene fragment was directly cloned into the expression vector pGEX-4T-1. The resulting pGEX-4T-VP1M plasmid was transformed into BL21 （DE3） and expression of VP1M was induced with IPTG. The recombinant VP1M was harvested in inclusion body form and visualized in Western blot analysis. In addition, an indirect ELISA was developed for detecting antibodies against DHAV-1 using the recombinant VP1M protein. The results showed that the assay was specific, sensitive and reproducible, which could be used as a serological method for rapid diagnosis, epidemiology and surveillance of DHAV-1.
出处
《中国动物传染病学报》
CAS
北大核心
2015年第2期7-14,共8页
Chinese Journal of Animal Infectious Diseases
基金
山东出入境检验检疫局基金项目(SK201301)
青岛市公共领域支撑计划(12-1-3-80-jh)
国家自然科学基金项目(31101848)
