基于ORF2蛋白的山羊星状病毒抗体间接ELISA检测方法的建立与应用  

Establishment and Application of Antibody Indirect ELISA Detection Method Based on Caprine Astrovirus ORF2 Protein

作　　者：于皓同 马永杰 王凯茸 丁雨欣 张淑霞[1] 张琪[1] 许信刚[1] YU Hao-tong;MA Yong-jie;WANG Kai-rong;DING Yu-xin;ZHANG Shu-xia;ZHANG Qi;XU Xin-gang(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《动物医学进展》2025年第3期17-22,共6页Progress In Veterinary Medicine

基　　金：国家重点研发计划项目(2021YFD1600704);陕西省农业专项资金项目(XNDY2210)。

摘　　要：为了建立山羊星状病毒(Caprine astrovirus)的血清抗体检测方法,从经RT-PCR检测星状病毒阳性的腹泻羊粪便中克隆山羊星状病毒ORF2基因并构建表达载体,用原核表达技术制备重组ORF2蛋白,重组蛋白纯化复性后作为包被抗原,建立山羊星状病毒血清抗体间接ELISA检测方法,并对临床收集的山羊血清样本进行检测。结果显示,ORF2重组蛋白大小为42 ku,Western blot证明其具有良好的反应原性;ELISA检测方法最佳工作条件为6μg/mL抗原包被酶标板,一抗血清稀释倍数为1∶100,酶标二抗稀释倍数为1∶5000,阴阳临界值为0.327。用该方法对山羊流产衣原体、立克次体、山羊支原体山羊肺炎亚种及伪结核棒状杆菌的阳性血清进行检测,结果均为阴性;当阳性血清稀释至1024倍时检测结果仍为山羊星状病毒抗体阳性;批内、批间变异系数均小于10%。用该方法对陕西省部分羊场收集到的288份血清样本进行检测,山羊星状病毒抗体阳性率为59.03%。建立了一种检测山羊星状病毒抗体的间接ELISA检测方法,为山羊星状病毒感染的诊断和流行病学调查提供了技术支持。In order to establish the method for the detection of caprine astrovirus serum antibody,the ORF2 gene of caprine astrovirus was cloned from diarrhea stools detected positive for astrovirus by RT-PCR and the expression vector was constructed.The recombinant ORF2 protein was prepared by prokaryotic expression technique.The recombinant protein was purified and refolded as the coated antigen,and an indirect ELISA method for detection of caprine astrovirus serum antibodies was established.The clinically collected caprine serum samples were tested by this method.The results indicated that the size of the expressed recombinant ORF2 protein is 42 ku.Western blot indicated that the recombinant protein had good reactivity.The optimal working conditions of the ELISA were 6μg/mL of antigen coated with enzyme-labeled plate,the dilution of first antiserum was 1∶100,the dilution of enzyme-conjugated secondary antibody was 1∶5000.The critical value of negative and positive samples was 0.327.In this method,the positive sera of Chlamydia abortus,Rickettsia,Mycoplasma capricolum subsp.capripneumoniae and Corynebacterium pseudotuberculosis were negative;When the positive serum was diluted to 1024 times,the test result was still positive for caprine astrovirus.The coefficient of variation of intra and interbatches was less than 10%.Using this method,288 serum samples collected from some goat farms in the Shaanxi provinces were detected,the positive rate of caprine astrovirus was 59.03%.This study established an indirect ELISA for detecting antibodies to caprine astrovirus,which provided technical support for diagnosis and epidemiological investigation of caprine astrovirus.

关 键 词：山羊星状病毒 ORF2蛋白 原核表达 间接酶联免疫吸附试验 

分 类 号：S852.659.6[农业科学—基础兽医学] S858.27[农业科学—兽医学]