鸭源鸡杆菌毒素蛋白GtxA表达和间接ELISA方法的建立及初步应用  

Expression of G.anatistoxin GtxA and development of an indirect ELISA for the detection of serum antibody against GtxA in chickens

作　　者：李欢[1] 杨霞[1] 赵军[1] 魏亚鹏[1] 陈陆[1] 王新卫[1] 常洪涛[1] 李永涛[1] 刘红英[1] 王川庆[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2015年第1期50-56,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31302090)

摘　　要：扩增鸡杆菌毒力基因gtxA全长序列,对GtxA蛋白的抗原性进行预测。将gtxA基因的部分抗原表位区所处片段克隆至pGEX-6P-1载体,在大肠杆菌BL21(DE3)细胞内表达。重组菌在37℃条件下经IPTG诱导表达,SDS-PAGE结果表明该蛋白高效表达,表达蛋白相对分子质量约为50 000,以可溶性蛋白和包涵体蛋白2种形式存在。Western blotting分析表明,获得的蛋白具有良好的反应原性和特异性。以纯化的GtxA蛋白为包被抗原建立了抗GtxA间接ELISA方法:抗原最佳包被质量为0.403μg/孔,一抗血清的最佳稀释度为1∶160,酶标二抗的最佳工作浓度为1∶2 000,阳性判定标准为血清D450≥0.28。应用建立的检测方法对680份临诊鸡血清进行检测,结果显示河南省不同地区鸡群鸡杆菌感染阳性率为19.06%(129/680)。结果表明,该方法可有效检测鸡杆菌血清抗体。Gallibacteriumis an infectious pathogen in chickens and other avian species,which is a significant cause of salpingitis and peritonitis,the representative species is Gallibacterium anatis.The partial gtxA gene was amplified by PCR and inserted into the expression vector pGEX-6P-1.The plasmid pGEX-6P-1-galli was transformed into E.coli BL21(DE3).The infusion protein was expressed when induced by IPTG.The molecular weight of recombinant protein was about 50 000 analyzed by SDS-PAGE,and the immunoreaction activity of the recombinant protein was confirmed by Western blotting.An indirect ELISA for detecting antibody against Gallibacterium anatis was developed by using expressed GtxA protein as coating antigen.The results showed that the optimal coating antigen was 0.403μg/well,the optimal dilution of the serum was 1∶160and the HRP-labeled rabbit anti-chicken IgG was 1∶2 000.The determine criteria of the indirect ELISA was samples D450value≥0.28 as positive.Chicken serum samples from different herds in Henan province were examined with the ELISA developed in present study and the result was compared with that of previously reported ELISA by using Gallibacteriumcell lysates as a coating antigen.The total positive rate of samples detected was 19.06%(129/680).The result showed that the methods established was specific,sensitive and can be used for clinical detection as well as epidemiology survey of Gallibacterium.

关 键 词：鸡杆菌 GtxA 融合蛋白 间接ELISA 抗体监测 

分 类 号：S852.61[农业科学—基础兽医学]