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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 and purify Mycobacterium tuberculosis (M.tb) heparin-binding haemagglutinin adhesion (HBHA) in prokaryotic cells and evaluate its value in serodiagnosis of tuberculosis. Methods The hbha gene was amplified from the M.tb H37Rv genome by PCR and cloned into the prokaryotic expression vector p ET-28a (+). The recombinant plasmid p ET-28ah was constructed and transformed into E. coli BL21 (DE3) expression. The expressed recombinant HBHA (r HBHA) protein was purified by Ni2 + affinity chromatography and its antigen specificity was analyzed by Western blot. Serum samples of 71 confirmed TB patients and 30 healthy controls were collected. Serum anti-HBHA Ig A and Ig G antibody levels were measured by ELISA. ESAT-6 and Ag85A were used as control antigens to evaluate the effect of r HBHA on tuberculosis serology Diagnostic value. Results The recombinant plasmid p ET-28ah was confirmed by restriction enzyme digestion and sequencing. The recombinant protein was expressed mainly in the form of inclusion bodies. High purity r HBHA protein was obtained after purified by Ni2 + affinity chromatography. The purified r HBHA The specific anti-His-tag IgA levels in serum of patients with tuberculosis (TB) were significantly higher than those of healthy controls (HC) (P <0.05). The level of r HBHA antigen-specific IgA in TB group was also significantly higher than that in HC group (P <0.05). There was no significant difference in ESA-6 and Ag85A antigen-specific IgA levels between the two groups P> 0.05). The ROC curve analysis showed that the diagnostic efficiency of r HBHA antigen in detecting Ig A was better, the area under the curve was 0.711. The Ig A diagnostic efficacy of the other two control antigens ESAT-6 and Ag85A was normal. The area under the curve was 0.512 and 0.531. Conclusion The diagnostic efficacy of HBHA antigens for tuberculosis is good and can be used as one of the candidate antigens for serodiagnosis of tuberculosis.