Preparation Of Mycobacterium Heparin-binding Haemagglutinin And Its Application

BackgroundTuberculosis(TB) is one of the greatest public health challenges in the world and remains the leading cause of death due to a single infectious diseases. The traditional bacteriological methods need a long time or only have a poor positive detection rate for pulmonary TB especially extrapulmonary TB diagnosis. TB therapy also face a big problem because of a global emergence of drug-resistant strains and relative relapse in drug development. Mycobacterium bovis bacillus Calmette-Guerin(BCG), a solely used TB preventive vaccine, can not effectively prevent occurrence of adult TB. Hence, the development of new diagnostic, prophylactic and therapeutic strategies are required for the control of TB.M. tuberculosis produces on its surface modified protein antigens such as Mycobacterium heparin-binding haemagglutinin (HBHA). HBHA is a principal mycobacterial adhesin for epithelial cells, and is critical for escaping from the lungs and thus for extrapulmonary dissemination. At present, the study of mycobacterial adhesins are focused on the relationshipbetween adhesins and host cells. It is only the beginning about application of mycobacterial adhesin in the diagnosis, treatment and prevention of TB. It is significant necessity that explore the function of mycobacterial adhesin HBHA in immune response of TB and its application in the diagnosis, treatment and prevention of TB.Purpose and methods1 To select specific primers of 16S rDNA and hbhA coding gene of mycobacterium for amplifying appropriate gene fragment, and observe whether there is hbhA gene deletion among the standard strains of Mycobacterium tuberculosis and nontuberculos mycobacterium, the clinical isolates of pulmonary and extrapulmonary tuberculosis.2 HBHA was purified from mycobacterium bovis BCG by heparin-Sepharose chromatography, and hbhA gene cloned into E. coli for expression of HBHA respectively. HBHA was injected into mouse and observe production of anti-HBHA antibody and its immunogenicity.3 To select pulmonary and extrapulmonary tuberculosis patients, PPD(+) and PPD(—) healthy control, and detect serum anti-HBHA antibody levels by ELISA in different groups. At the same time, we also want to know whether HBHA can play a role in immunological prevention and treatment in animal tuberculosis.Results1 All standard strains of M. tuberculosis have hbhA gene, and all other mycobacterium are negative by PCR experiments. Among 3 pulmonary TB patients isolate strains are positive in PCR amplification, and among selected 5 extrapulmonary TB isolate patients strains , 3 samples are positive and other 2 samples are negative in amplification of hbhA gene. We analysis 2 PCR negative strains by 16 S rDNA sequencing that one is M. fortuitum, the other is other bacterium.2 HBHA protein can increase antibody titer 100,000 times of immunized mouse compared with control group. It indicates that HBHA have good immunogenicity. Serum antibody level by ELISA can distinguish TB patients and healthy control, but can not distinguish PPD(+) and PPD(—) healthy control groups effectively. Native HBHA have a good sensitivity and specificity in TB immunological diagnosis compared with recombinant HBHA.Conclusions1 The hbhA gene of M tuberculosis have been cloned into PET-32a(+) expression vector, and got a high expression in E. coli. The recombinant HBHA was accumulated as inclusion bodies mainly in E. coli. The inclusion bodies were purified and acquired abundant HBHA protein.2 Native HBHA protein were acquired from culture filters of M.