弓形虫GRA6-GRA2蛋白表达及检测应用

Protein Expression of GRA6-GRA2 of Toxoplasma gondii and Its Application for Test Strip Detection Method

为建立一种敏感性好、特异性强、方便快捷的检测猫弓形虫抗体的方法,本研究通过分子生物学方法、优化合成了GRA1-GRA7、SAG1和GRA6-GRA2基因,采用大肠杆菌ER2566表达了GRA1-GRA7、SAG1和GRA6-GRA2三种蛋白;表达产物利用His亲和层析柱纯化后,经Western blot鉴定三种蛋白表达产物都有较好的抗原特异性;通过ELISA方法进行检测比较,确定GRA6-GRA2蛋白敏感性最高;以GRA6-GRA2蛋白作为标记抗原,制备弓形虫抗体检测试纸条,分别对敏感性对照阳性血清、特异性对照血清进行检测,并与弓形虫抗体间接血凝法对289份临床血清进行对比检测。结果表明:检测敏感性对照阳性血清可检测至1∶256,检测临床常见的猫细小病毒阳性血清、猫杯状病毒阳性血清和猫疱疹病毒阳性血清结果均为阴性;在弓形虫抗体检测试纸条和弓形虫抗体间接血凝法对289份临床血清的对比检测中,阳性率分别为2.42%和2.07%,符合率为99.5%。通过以上结果可知,采用GRA6-GRA2蛋白作为诊断抗原制备的胶体金试纸,具有良好的敏感性和特异性,为弓形虫病的快速诊断提供了切实可行的检测手段。

This study developed a sensitive,specific and convenient method for the detection of toxoplasma antibody in cats.In this study,genes of GRA1-GRA7,SAG1 and GRA6-GRA2 were optimized and synthesized by molecular biological methods.Three proteins of GRA1-GRA7,SAG1 and GRA6-GRA2 were expressed in E.coli ER2566.The expressed products were purified using his-affinity chromatography column and identified in Western blot with good antigen specificity.The GRA6-GRA2 protein showed the highest sensitivity as determined by ELISA.Therefore,GRA6-GRA2 protein was used to prepare toxoplasma antibody detection test strips for detection of positive serum samples.Total 289 clinical serum samples were tested using these test strips and indirect hemagglutination(IHA)and the positive rates were 2.42%and 2.07%.The test strips showed detectable titer of positive serum at 1:256 but no reaction to clinically common feline parvovirus positive sera,feline calicivirus positive sera and feline herpes virus positive sera.The test strips with GRA6-GRA2 protein had good sensitivity and specificity,which might provide a feasible detection method for the rapid diagnosis of toxoplasmosis.