结核分枝杆菌肝素结合血凝黏附素的原核表达及其在结核病血清学诊断中的应用  

Prokaryotic expression of heparin-binding haemagglutinin adhesin from Mycobacterium tuberculosis and its application in serological diagnosis of tuberculosis

作　　者：问寒利 李海聪[2] 李根[1] 赵慧敏[2] 施理敏 韩伟靖 吴康[2] 范小勇[1] 

机构地区：[1]温州医科大学检验医学院生命科学学院,浙江温州325035 [2]上海市(复旦大学附属)公共卫生临床中心,上海201508

出　　处：《中国生物制品学杂志》2016年第5期497-502,共6页Chinese Journal of Biologicals

基　　金：国家"十二五"重大传染病专项(2013ZX10003007-003);国家自然科学基金项目(81273328;31170876);上海市科委医学引导类项目(134119a5200);上海市卫生局级课题青年项目(20124Y090)

摘　　要：目的原核表达与纯化结核分枝杆菌(Mycobacterium tuberculosis,M.tb)肝素结合血凝黏附素(heparin-binding haemagglutinin adhesion,HBHA),并初步评估其在结核病血清学诊断中的价值。方法采用PCR法从M.tb H37Rv基因组中扩增hbha基因,克隆至原核表达载体p ET-28a(+),构建重组表达质粒p ET-28ah,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的重组HBHA(r HBHA)蛋白经Ni2+亲和层析柱纯化后,Western blot法分析其抗原特异性。收集71例确诊的结核病患者和30名健康体检者血清,采用ELISA法检测血清中的抗HBHA Ig A、Ig G抗体水平,并以ESAT-6、Ag85A作为对照抗原,评估r HBHA在结核病血清学诊断中的价值。结果重组表达质粒p ET-28ah经酶切及测序证实构建正确;表达的重组蛋白主要以包涵体形式存在,经Ni2+亲和层析柱纯化,可得到高纯度的r HBHA蛋白;纯化的r HBHA蛋白可与小鼠抗His-tag单克隆抗体特异性结合;结核病患者(TB)组血清中抗r HBHA、ESAT-6、Ag85A抗原特异性Ig G水平均显著高于健康对照(HC)组(P<0.05),且TB组的r HBHA抗原特异性Ig A水平也显著高于HC组(P<0.05),而两组间ESAT-6、Ag85A抗原特异性Ig A水平差异无统计学意义(P>0.05);ROC曲线分析显示,r HBHA抗原检测Ig A的诊断效能较好,曲线下面积为0.711,其他两种对照抗原ESAT-6、Ag85A的Ig A诊断效能一般,曲线下面积分别为0.512和0.531。结论 r HBHA抗原用于结核病的诊断效能较好,可作为结核病血清学诊断的备选抗原之一。Objective To express heparin-binding haemagglutinin adhesin（HBHA）from Mycobacterium tuberculosis（M.tb）in prokaryotic cells,purify the recombinant protein and evaluate its significance in serological diagnosis of tuberculosis（TB）.Methods The hbha gene was amplified from the genome of M.tb H37 Rv by PCR and cloned into prokaryotic expression vector p ET-28a（＋）.The constructed recombinant plasmid p ET-28 ah was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed recombinant HBHA（r HBHA）was purified by nickel ion affinity chromatography and analyzed for antigenic specificity by Western blot.Serum samples were collected from 71 patients with TB and 30 healthy volunteers,and determined for Ig A and Ig G levels against HBHA by ELISA,based on which the significance of r HBHA in serological diagnosis of TB was evaluated using ESAT-6 and Ag85 A as control antigens.Results Both restriction analysis and sequencing proved that recombinant plasmid p ET-28 ah was constructed correctly.The expressed recombinant HBHA mainly existed in a form of inclusion body,and reached a high purity after purification by nickel ion affinity chromatography.The purified HBHA showed specific binding to mouse anti-His tag monoclonal antibody.The Ig G levels against r HBHA,ESAT-6 and Ag85 A as well as Ig A level against r HBHA in patients with TB were significantly higher than those in healthy volunteers as control（P〈.05）.However,the Ig A levels against ESAT-6 and Ag85 A in two groups showed no significant difference（P〈.05）.Receiver operator characteristic（ROC）curve showed good diagnostic effect of r HBHA-specific Ig A assay with AUC of 0.711,which was superior to those of ESAT-6（0.512）and Ag85A（0.531）.Conclusion Recombinant HBHA was showed good efficiency in diagnosis of TB,which might be used as a candidate antigen for serological diagnosis of the disease.

关 键 词：结核分枝杆菌 肝素结合血凝黏附素 免疫学诊断 

分 类 号：R378.911[医药卫生—病原生物学] R446.61[医药卫生—基础医学]