基于TPX和CatB4重组蛋白的绵羊肝片吸虫病间接ELISA诊断方法的建立  

Establishment of indirect ELISA for diagnosis of sheep fascioliasis hepatica based on TPX and CatB4 recombinant proteins

作　　者：魏雅婷 孙益春 郑芦珊 杨莹 张金鹏 庞紫璇 路义鑫[1] WEI Ya-ting;SUN Yi-chun;ZHENG Lu-shan;YANG Ying;ZHANG Jin-peng;PANG Zi-xuan;LU Yi-xin(Heilongjiang Key Laboratory for Zoonosis/College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学动物医学学院黑龙江省动物源性人兽共患病重点实验室,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2021年第5期581-587,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFD0501200)。

摘　　要：为了建立早期快速诊断绵羊肝片吸虫病的血清学方法,原核表达肝片吸虫TPX和CatB4重组蛋白。分别以TPX、CatB4重组蛋白作为包被抗原,建立2种检测肝片吸虫血清抗体的间接ELISA方法。采用棋盘式方法优化反应的最佳血清稀释度和抗原包被量,同时对间接ELISA方法的其他反应条件进行了筛选。蛋白免疫印迹结果显示,重组蛋白具有良好的抗原活性。这2种间接ELISA方法批内和批间重复试验的最大变异系数均小于10%,且这2种方法与华支睾吸虫、日本血吸虫和捻转血矛线虫阳性血清均无交叉反应。对来自黑龙江省各地区的95份绵羊血清进行检测,阳性率分别为18.95%和22.11%。结果表明,本研究中建立的2种间接ELISA方法具有良好的特异性和敏感性,为肝片吸虫病的早期诊断和流行病学调查提供了必要手段。In order to establish a serological method for early and rapid diagnosis of sheep fascioliasis hepatica,Fasciola hepatica TPX and CatB4 recombinant proteins were expressed by the prokaryotic expression.The TPX and Cat B4 recombinant proteins were used as coating antigens to establish two kinds of indirect ELISA,which were detected for serum antibodies to F.hepatica.The checkerboard method was used to optimize the optimal serum dilution and antigen coating amount,at the same time,other reaction conditions of the methods were screened. Western-blotting results showed that the recombinant proteins had good antigenic activity.The maximum coefficient of variation of the two indirect ELISA methods within and between repeated experiments was less than 10%,and the two methods had no cross-reactivity with positive serum to Clonorchis sinensis,Schistosoma japonicum and Haemonchus contortus respectively.The positive rates of 95 sheep serum samples from Heilongjiang Province detected by the two methods were 18.95% and 22.11% respectively.The results showed that the two indirect ELISA methods established in this study had good specificity and sensitivity,and provide necessary means for early diagnosis and epidemiological investigation of fascioliasis hepatica.

关 键 词：肝片吸虫 硫氧还蛋白过氧化物酶 组织蛋白酶B4 间接ELISA 

分 类 号：S852.735[农业科学—基础兽医学]