猪瘟病毒E0蛋白的原核表达及其间接ELISA方法的建立  

Prokaryotic Expression of CSFV E0 Protein and Development of an Indirect ELISA for Detection of Antibody

作　　者：陈振海[1] 王琴[2] 范学政[2] 宁宜宝[2] 王在时[2] 沈青春[2] 夏春[1] 

机构地区：[1]中国农业大学动物医学院,北京100094 [2]中国兽医药品监察所国家猪瘟参考实验室,北京100081

出　　处：《中国病毒学》2005年第2期135-139,共5页Virologica Sinica

基　　金：国家"十五"科技攻关子专题(2002BA514A 18 2);国家自然科学基金项目(30270984)

摘　　要：表达猪瘟病毒保护性抗原E0蛋白用以建立猪瘟抗体诊断方法,在标记疫苗(Marker vaccine)的研制和应用上具有重要的血清学鉴别功能,对于监测猪瘟病毒的流行情况和疫苗免疫情况及制定免疫程序具有重要作用。将猪瘟兔化弱毒株(HCLV)E0 基因分别插入原核表达质粒pGEX4T, pET30, pMAL p2X 中,并分别以BL21 和BL21 codonplus(DE3) RP, BL21(DE3)和BL21 codonplus(DE3) RP,TB1 和BL21 codonplus(DE3) RP为表达菌株。通过摸索IPTG诱导浓度,诱导温度,菌体收获时间,确定E0 基因能在pGEX4T/ BL21 codonplus(DE3) RP和pMAL p2X/ BL21 codonplus(DE3) RP中获得高效表达,表达产量分别占菌体总蛋白的15%和30%,表达的重组蛋白主要为包涵体形式。分别采用B per试剂和超声波裂解配以曲通尿素对包涵体进行洗涤两种方法在pGEX表达系中取得了较好的效果。使用分步透析法对变性的包涵体进行复性,将复性蛋白过GST亲和层析柱得到纯化的GST E0融合蛋白。以GST E0融合蛋白为诊断抗原,通过摸索抗原包被浓度,抗体血清稀释倍数初步建立了用间接ELISA检测猪瘟血清E0抗体的方法,为进一步开发猪瘟抗体检测试剂盒奠定基础。E0 protein of Classical swine fever virus(CSFV) is of discriminatory feature in serum diagnostic test for developing CSFV Marker vaccine. It is also expected to be useful for monitoring CSFV prevalence,evaluating immunization efficacy and establishing immunization procedure. To obtain a large amount of E0 protein in vitro ,the Hog cholera lapinised virus(HCLV) E0 gene was cloned into prokaryotic expression plasmids, pGEX4T、pET30 and pMAL-p2X respectively.After the recombinant expression plasmids were transformed into BL21、BL21(DE3)、TB1、BL21-codonplus(DE3)-RP respectively, the expression product were analyzed by investigating the IPTG induction concentration ,incubation temperature and time. The results revealed that the E0 protein could be expressed in the systems, namely pGEX4T/BL21-codonplus(DE3)-RP and pMAL-p2X/ BL21-codonplus(DE3)-RP as inclusion bodies accounted for 15% and 30% of whole bacterial protein. Two methods , B-per reagent and triton-urine, were applied to wash the inclusion body and both access to the relatively good effect. After the protein refolding of denaturant inclusion body following dialysis, we got the pure recombinant GST-E0 protein by GST affinity columns. Using the purified protein as coating antigen, an indirect ELISA is developed for detecting the anti-E0 antibody in the CSFV serum by exploring the concentration of coating antigen and dilution degree of serum, which will provide a good fundament for development of CSFV antibody surveillance kit.

关 键 词：猪瘟兔化弱毒 EO基因 原核表达 包涵体 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]