结核分枝杆菌黏附素HBHA在结核病免疫学诊断中的应用

目的 探讨结核分枝杆菌肝素结合血凝黏附素(Heparin-binding haemagglutinin adhesin,HBHA)在结核病免疫诊断方面的应用价值.方法 将在苏通培养基中培养至稳定期的卡介苗菌株(BCG)菌体超声裂解,离心后取上清并通过CL-6B层析柱,利用含0～500 mmol/L氯化钠的磷酸盐缓冲溶液(PBS)进行梯度洗脱后获得天然HBHA.以BCG基因组为模板PCR扩增结核分枝杆菌hbha基因片段,质粒pET-32a为载体,大肠杆菌作为宿主菌制备原核表达的HBHA重组蛋白.以纯化的天然HBHA蛋白和原核表达的HBHA蛋白为包被抗原,分别对肺结核组、肺外结核组、PPD阳性和PPD阴性健康对照组各47例血清通过ELISA方法检测抗HBHA的抗体水平.应用SPSS 11.5统计软件对各研究组数据进行方差齐性检验,然后进行t检验,并计算各研究组的敏感度和特异度.结果 含有375 mmol/L氯化钠的PBS洗脱液可以洗脱获得较纯的天然HBHA蛋白.利用重组HBHA所带的组氨酸标签进行特异分离、纯化.ELISA检测结果表明天然HBHA蛋白和重组HBHA蛋白、PPD阳性和PPD阴性健康对照组之间血清抗体水平差异不明显.肺结核组与肺外结核组的血清抗体ELISA检测的吸光度值在散点图中分布有明显差异,肺结核组吸光度值集中分布在0.30～0.40之间,肺外结核组吸光度值分布在0.35～0.45之间,经统计学检验结果差异具有统计学意义(t=12.224,P＜0.05).结核组(肺结核和肺外结核)与对照组(PPD阴性,PPD阳性)吸光度值分布具有显著不同,对照组全部小于0.30,结核组吸光度值则集中分布于0.30～0.45之间.经统计学检验(t=25.909,P＜0.05)血清抗体水平具有显著性差异.结论 结核分枝杆菌黏附素HBHA在结核病免疫学诊断的应用中具有较好的特异度和敏感度,有望用于结核病、尤其是肺外结核病的实验室辅助诊断.

Purification of the heparin-binding haemagglutinin adhesin of Mycobacterium tuberculosis and its application in diagnosis of tuberculosis

Objective To investigate the application of heparin-binding haemagglutinin adhesin (HBHA) in tuberculosis (TB) diagnosis.Methods We prepared native HBHA from cultivated Mycobacterium Boris Calmetta Guerin (BCG) in Suton liquid medium.After BCG grew to the stationary status.native HBHA was acquired by specific CL-6B chromatography column binding heparin.At the same time.we cloned hbhA gene from Mycobacterium tuberculosis into PET-32α (+) expression vector.Recombinant HBHA from E. coli was obtained.Based on the native HBHA and recombinant HBHA.we chose 4 groups of pulmonary TB,extra-pulmonary TB,PPD(-)and PPD(+)healthy control with 47 in each group and conducted ELISA from serum for specific HBHA antibody level.At last we calculated the sensitivity and specificity in TB diagnosis by detection of anti-HBHA antibody level.Results The native HBHA could be diluted and purified with the PBS containing the 375 mmol/L NaCl by specific CL-6B chromatography column binding heparin:There was no significant difierence in experimental result based on the natural and recombinant HBHA protein.also no difference between PPD (-) and PPD (+) healthy control groups.Serum antibody level by ELISA could distinguish pulmonary TB and ertra-pulmonary TB ( t=12.224,P＜0.05).The antibody level of the TB groups (pulmonary TB and ertra-pulmonary TB)was higher than the healthy control groups PPD (+) and PPD(-) healthy control] (t=25.909,P＜0.05).Conclusion Both recombinant and native HBHA can be used as immunological diagnosis in TB.It can be used in TB and especially extra-pulmonary TB diagnosis.