基于重组致密颗粒蛋白11的弓形虫胶体金免疫层析检测方法的建立  

Establishment of Toxoplasma gondii Colloidal Gold Immunochromatographic Detection Method Based on Recombinant Dense Granular Protein 11

作　　者：张艳玲 孟庆玲[1] 乔军[1] 任宏琪 赵春光[1] 宁程程 季春晖 才学鹏[2] ZHANG Yan-ling;MENG Qing-ling;QIAO Jun;REN Hong-qi;ZHAO Chun-guang;NING Cheng-cheng;JI Chun-hui;CAI Xue-peng(College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang 832003,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou,Gansu 730046,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《动物医学进展》2021年第3期12-16,共5页Progress In Veterinary Medicine

基　　金：“十三五”国家重点研发计划项目(2016YFD0501005)。

摘　　要：将弓形虫致密颗粒蛋白11(GRA11)部分基因片段插入pET32a(+)载体,构建原核表达载体pET-32a(+)-GRA11,并转化至大肠埃希氏菌BL21(DE3)中进行诱导表达,用SDS-PAGE和Western blot对诱导表达的重组蛋白GRA11 (rGRA11)进行反应原性鉴定。以rGRA11为检测线,兔抗犬IgG为质控线,制备胶体金免疫层析试纸条,与商品化的弓形虫抗体检测试剂盒对119份临床血清样品进行检测对比试验。成功构建了pET-32a(+)-GRA11原核表达载体,SDS-PAGE检测到的蛋白大小约36 ku。Western blot结果表明,弓形虫rGRA11蛋白可被弓形虫阳性血清所识别,具有较强的反应原性。与商品化弓形虫抗体间接血凝试剂盒相比,建立的免疫胶体金层析法敏感性为81.25%,特异性为100%,两种方法的总符合率为97.4%,为弓形虫病临床诊断方法研究奠定基础。Part of the gene fragment of Toxoplasma gondii dense granular protein 11(GRA11)was inserted into pET32a(+)vector to construct prokaryotic expression vector pET-32a(+)-GRA11,and transformed into E.coli BL21(DE3)for induced expression,SDS-PAGE and Western-blot were used to identify the reactogenicity of the recombinant protein GRA11(rGRA11).Using rGRA11 as the detection line and rabbit anti-canine IgG as the quality control line,a colloidal gold immunochromatographic test strip was prepared,and 119 clinical serum samples were compared with commercial toxoplasma antibody detection kits.The results showed that the pET-32a(+)-GRA11 prokaryotic expression vector was successfully constructed.The size of the protein detected by SDS-PAGE was about 36 ku.Western blot results showed that the Toxoplasma gondii rGRA11 protein can be recognized by Toxoplasma gondii positive sera and has a strong reactogenicity.Compared with the commercial Toxoplasma gondii antibody indirect hemagglutination kit,the established immunocolloid gold chromatography method has a sensitivity of 81.25%and a specificity of 100%.The total agreement rate of the two methods is 84.21%.The study provided a basis for research on clinical diagnostic methods of toxoplasmosis.

关 键 词：弓形虫 原核表达 胶体金免疫层析试纸条 

分 类 号：S852.71[农业科学—基础兽医学]