结核分枝杆菌Rv3425特异性蛋白片段与CFP10-ESAT6融合蛋白在结核抗体检测中的应用价值  

Diagnostic Value of Specific Protein Fragment RV3425 and Fusion Protein CFP10-ESAT6 in Tuberculosis Antibody Detection

作　　者：吴静希[1,2] 岳俊杰[3] 姜永强[2] 郝淮杰[2] 李艳[1] 

机构地区：[1]军事医学科学院附属医院,北京100071 [2]军事医学科学院微生物流行病研究所,北京100071 [3]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2011年第3期394-397,423,共5页Letters in Biotechnology

基　　金：首都特色临床医学技术发展研究课题(Z08050703080801)

摘　　要：目的:对结核分枝杆菌Rv3425蛋白进行生物信息学分析及抗原表位预测,评价Rv342519-176重组蛋白与CFP10-ESAT6融合蛋白在结核抗体检测中的应用。方法:PCR扩增结核杆菌Rv3425蛋白19～176位的编码基因片段,原核表达并纯化Rv342519-176重组蛋白(rRv342519-176)和CFP10-ESAT6融合蛋白(rCFP10-ESAT6),建立以重组蛋白为抗原的ELISA方法,评价2种蛋白在结核抗体检测中的联合应用价值。结果:具有抗原表位的rRv342519-176与rCFP10-ESAT6在大肠杆菌中高效表达;ELISA结果显示,rRv342519-176的敏感性为39%,特异性为97.5%;rCFP10-ESAT6的敏感性为37%,特异性为95%;2种蛋白联合检测,敏感性提高到57%,特异性为95%。结论:生物信息学预测Rv3425的优势抗原表位,表达并纯化的rRv342519-176和rCFP10-ESAT6在结核抗体检测中具有一定的抗原互补性,在保持特异性的基础上可显著提高检测敏感性。Objective：Bioinformatics analysis and epitope prediction of Rv3425 in Mycobacterium tuberculosis.Evaluating the diagnostic value of Rv342519-176 recombinant protein and CFP10-ESAT6 fusion protein in tuberculosis（TB）.Methods：The gene coding 19～176 amino acid fragment of Rv3425 was amplified from Mycobacterium tuberculosis genome by PCR.Prokaryotic expression vector of Rv342519-176 was constructed and recombinant protein（rRv342519-176） and CFP10-ESAT6 fusion protein（rCFP10-ESAT6） were expressed and purified.Establishing ELISA testing method with recombinant protein and fusion protein for antigen,and assessing the combined value of two proteins in TB-antibody detection.Results：The rRv342519-176 protein fragment with dominant epitope and rCFP10-ESAT6 were expressed effectively in E.coli.The ELISA result showed certain sensibility（39%） and high specificity（97.5%）,when rRv342519-167 was antigen for clinical diagnosis in tuberculosis.The sensibility and specificity were respectively 37% and 95%,when rCFP10-ESAT6 was antigen.Furthermore,two proteins conjoint analysis by ELISA showed higher sensibility（57%） and specificity（95%）.Conclusion：The epitope of Rv3425 was predicted by bioinformatics.The expressed and purified rRv342519-167 and rCFP10-ESAT6 had a certain complementary antigen in TB-antibody detection,and provided a reference with improving the diagnostic sensitivity in joint diagnostic antigen with keeping high specificity.

关 键 词：结核分枝杆菌 Rv3425蛋白 融合蛋白CFP10-ESAT6 抗原性 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]