以重组融合蛋白为基础的钩端螺旋体酶联免疫吸附试验检测试剂盒的初步研制  

Preliminary development of an enzyme-linked immunosorbent assay kit based on recombinant fusion antigen protein for detection of Leptospira

作　　者：胡玉山[1] 刘巧谊[1] 肖丽红[1] 周勇[1] 侯水平[1] 刘俊华[1] HU Yu-shan;LIU Qiao-yi;XIAO Li-hong;ZHOU Yong;HOU Shui-ping;LIU Jun-hua(Guangzhou Center for Disease Control and Prevention,Guangzhou,Guangdong 510440,China)

机构地区：[1]广州市疾病预防控制中心

出　　处：《实用预防医学》2019年第7期808-811,共4页Practical Preventive Medicine

基　　金：广州市科技计划项目(2014J4100074);广州市医学重点学科建设项目(2017-2019-07)

摘　　要：目的建立以钩端螺旋体融合蛋白LipL32-LipL41(LipL32:脂蛋白L32,lipoprotein32;LipL41:脂蛋白L41,lipoprotein41)为包被抗原的酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测试剂。方法选择问号钩端螺旋体LipL32和LipL41作为研究靶标,采用连接引物PCR法构建lipL32-lipL41融合基因,原核表达获得重组蛋白,以该蛋白为包被抗原建立ELISA检测体系,对检测体系的灵敏度、特异性、稳定性等性能指标进行评价。结果扩增获得了1 900 bp左右的lipL32-lipL41融合基因,原核表达出了90 kDa左右的重组融合蛋白LipL32-LipL41,建立了基于LipL32- LipL41的ELISA检测体系,采用77例确诊病例和85例阴性对照血清进行初步验证,检测体系的灵敏度为96.1%,特异度为97.6%,粗一致性为96.9%,约登指数为0.937。结论以LipL32-LipL41重组融合蛋白为包被抗原建立ELISA免疫检测初步验证的灵敏度、特异性和稳定性较好,可进行下一步验证工作。Objective To establish an enzyme-linked immunosorbent assay (ELISA) detection reagent based on Leptospira fusion protein LipL32- LipL41 as a coated antigen.Methods Leptospira interrogans lipoprotein32 (LipL32) and lipoprotein41 (LipL41) were selected as research targets. The fusion gene lipL32-lipL41 was constructed by using PCR with ligation primers. The recombinant protein was expressed in prokaryotic expression system. The ELISA detection system was established by using the fusion protein as the coated antigen. The sensitivity, specificity and stability of the detection system were evaluated.Results About 1,900 bp lipL32-lipL41 fusion gene fragment was obtained. The molecular weight of the recombinant fusion protein LipL32-LipL41 prokaryotically expressed was about 90 kDa, and an ELISA detection system based on LipL32-LipL41 was established. 77 confirmed serum samples and 85 negative control ones were used for preliminary verification. The sensitivity, specificity, crude consistency and Yorden index of the detection system were 96.1%, 97.6%, 96.9% and 0.937 respectively.Conclusions It is preliminarily verified that the ELISA diagnostic kit based on the recombinant fusion protein LipL32-LipL41 as a coated antigen has high sensitivity, specificity and stability, and can be proceeded with the next verification.

关 键 词：钩端螺旋体 融合蛋白LipL32-LipL41 ELISA 

分 类 号：R514.4[医药卫生—内科学]