Zhang Xu-Xia, Sun Zhao-Gang, Li Yan, Zhang Jian-Yuan, Duanmu Hong-Jin, Li Chuan-You
Department of Bacteriology & Immunology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2008 Jun;31(6):448-52.
To investigate the application of heparin-binding haemagglutinin adhesin (HBHA) in tuberculosis (TB) diagnosis.
We prepared native HBHA from cultivated Mycobacterium Bovis 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-32alpha (+) 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.
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 difference 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).
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.
探讨肝素结合血凝素黏附素（HBHA）在结核病（TB）诊断中的应用。
我们在苏通液体培养基中培养卡介苗（BCG）以制备天然HBHA。待BCG生长至稳定期后，通过特定的CL-6B肝素亲和层析柱获得天然HBHA。同时，我们从结核分枝杆菌中克隆hbhA基因到PET-32alpha(+)表达载体中，获得来自大肠杆菌的重组HBHA。基于天然HBHA和重组HBHA，我们选取了4组肺结核、肺外结核、PPD（-）和PPD（+）健康对照，每组47例，检测血清中特异性HBHA抗体水平，进行酶联免疫吸附测定（ELISA）。最后通过检测抗HBHA抗体水平计算结核病诊断的敏感性和特异性。
天然HBHA可通过特定的CL-6B肝素亲和层析柱用含375 mmol/L氯化钠的磷酸盐缓冲盐水（PBS）进行稀释和纯化；基于天然和重组HBHA蛋白的实验结果无显著差异，PPD（-）和PPD（+）健康对照组之间也无差异。ELISA检测的血清抗体水平可区分肺结核和肺外结核（t = 12.224，P < 0.05）。结核病组（肺结核和肺外结核）的抗体水平高于健康对照组[PPD（+）和PPD（-）健康对照]（t = 25.909，P < 0.05）。
重组和天然HBHA均可用于结核病的免疫诊断。它可用于结核病尤其是肺外结核的诊断。
