隐孢子虫重组蛋白包被抗原的筛选及ELISA方法的建立及初步应用  

Development of an ELISA for detection of Cryptosporidium infection in cattle

作　　者：李军[1,2] 唐林生 彭昊[1,2,3] 陶立[1,2] 潘艳 陈泽祥[1,2] 许力干 谢永平[1,2] 杨威 黄维义[3] 

机构地区：[1]广西兽医研究所,广西南宁530001 [2]广西畜禽疫苗新技术重点实验室,广西南宁530001 [3]广西大学动物科学技术学院,广西南宁530005

出　　处：《中国预防兽医学报》2015年第4期286-290,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：公益性行业科研专项(201103008);广西基本科研业务费专项(桂科专项12-2);广西壮族自治区水产畜牧兽医局科技计划项目(桂渔牧科1304525)

摘　　要：为建立检测牛隐孢子虫抗体的间接ELISA方法,本研究将重组表达的隐孢子虫卵囊壁蛋白（COWP）、热休克蛋白70（HSP70）蛋白及COWP-HSP70串联蛋白分别作为包被抗原,进行反应条件的优化,建立了3种间接ELISA检测方法。建立的这3种方法均具有较高的特异性和敏感性。与伊氏锥虫,球虫和弓形虫阳性血清均无交叉反应,最低检出血清稀释倍数分别为1∶1 600、1∶3 200和1∶3 200。经过筛选,确定COWP-HSP70串联蛋白为包被抗原的间接ELISA方法为最佳检测方法。以该方法进行的重复性试验结果显示,其批内和批间变异系数分别为3.9%～8.2%和4.7%～8.3%。采用建立的ELISA方法与传统的粪检法分别对174份临床样品进行检测,隐孢子虫感染的检出率分别为9.2%和12.07%,两者符合率为97.13%。本研究建立的以COWPHSP70串联蛋白为包被抗原的牛隐孢子虫间接ELISA检测方法适用于临床牛隐孢子虫感染的检测。In order to develop method for the detection of Cryptosporidium infection in cattle, COWP, HSP70 and COWP-HSP70 fusion protein were evaluated as coating antigens for the indirect ELISA establishment. Results showed that all three proteins displayed high specificity and sensitivity as antigens to detect Cryptospoddium infection, and no cross-reactions were detected with any of other positive sera, such as the sara against Trypanosoma evansi, Emimeria spp and Toxoplasma gondii. The lowest detectable diluted titers of these three serums were 1：1,600, 1：3,200 and 1：3,200, respectively. Among them, COWP- HSP70 fusion protein had the highest affinity to the positive serum. The repetitive test of indirect ELISA coated with COWP- HSP70 showed that the intra- and inter-assay coefficient of variation were 3.9% to 8.2% and 4.7% to 8.3%, respectively. This method was applied to detect 174 clinic samples compared with routine fecal examination, and the positive rates were 9.2% and 12.07%, respectively. The coincidence rate of these two methods was 97.13%. Our data suggested that this method was potentially used to detect Cryptosporidium infection in cattle.

关 键 词：隐孢子虫 卵囊壁蛋白 热休克蛋白70 酶联免疫吸附试验 

分 类 号：S852.7[农业科学—基础兽医学]