蓝舌病病毒vp7基因的表达及其产物在c—ELISA中的应用

获得稳定、高效的具有良好抗原性的蓝舌病毒(Bluetongue virus，BTV)vp7基因重组抗原。将BTV编码群特异性抗原VP7的S7基因片段克隆至pMD18-T质粒载体中，构建S7克隆重组质粒，进行核苷酸序列分析。与已报道的多株BTV编码VP7的基因比较后发现，所测定毒株的核苷酸序列与BTV10型的S7基因同源性高达98．7％，推测的氨基酸同源性为99．3％，证实为BTV的S7基因。然后亚克隆插入pBAD/Thio TOPO表达载体，转化LGM194细胞，经抗性培养、PCR、限制性内切酶分析、测序鉴定，筛选获得BTV S7基因片段正向插入、有正确读码框的阳性克隆，成功构建了BTV群特异性抗原VP7的重组表达载体。经L-araboinose诱导表达，可稳定、高效地表达VP7蛋白抗原。SDS-PAGE、ELISA试验表明，表达蛋白为融合蛋白，具有反应原性，分子量约54．5kD，重组蛋白的获得率为1．52mg／g湿菌，其表达产量约占菌体总蛋白的12％左右，相当于93．5mg／L菌液。融合蛋白中含有BTV VP7特异性蛋白抗原，可作为c-ELISA包被抗原，为蓝舌病的免疫血清学诊断试剂的制备和分子生物学研究打下了坚实基础。

Expression of the Major Core Protein vp7 of Bluetongue Virus in E coli and Use as a Diagnostic Antigen in c-ELISA

The RNA S7 encoding serogroup-specific antigen VP7 of bluetongue virus (BTV) was cloned and inserted into pBAD/Thio TOPO vector. The recombinant plasmid was identified by restriction analysis, PCR and sequence. The results of SDS-PAGE and Western immunoblotting revealed that the VP7 was expressed in E coli LGM194 in a high level and the recombinant fusion protein with a molecular mass of approximately 54.5 kDa. Competition enzyme-linked immunosorbent assay (c-ELISA) indicated that the expressed BTV VP7 was similar in antigenicity to that of the native virus as revealed by its reactivity with monoclonal antibody (MAb) specific to BTV. The availability of a reliable BTV recombinant VP7 could enhance our existing assay for detection of BTV-specific antibodies. The performance of the c-ELISA and agar gel immunodiffusion (AGID) were compared using 3657 animal field sera from herds in China and animal imported. The purified recombinant VP7 antigen is highly immunogenic and has been shown in a c-ELISA to be reactive with antisera of 24 BTV serotypes (1 to 24). No cross-reactivities were detected between BTV and EHDV in c-ELISA tests. The data indicated that VP7 is a highly conserved protein amongst BTV serotypes. The c-ELISA tests indicate that the recombinant VP7 is useful as a diagnostic reagent for serological tests of BTV.