Identification Of The Adhesion Function Of Heparin-binding Hemagglutinin Protein From Mycobacterium Bovis

Bovine tuberculosis is one of the chronic consumptive zoonoses caused by Mycobacterium bovis which causes great economic losses for livestock industry and poses a serious threat to public health as well as food safety. HBHA protein is a surface protein encoded by the hbha gene in Mycobacterium bovis and is believed as one of the virulence factors. The adhesion ability of Mycobacterium bovis to cell is a prerequisite for the establishment of infection, and is one of the important indexes of its pathogenicity. Therefore, this study aimed to test the adhesion function of HBHA.1. The prokaryotic expression, polyclonal antibody preparation and adhesion determination of HBHA.The hbha gene of Mycobacterium bovis was amplified by PCR with Vallee III chromosomal DNA as template and then subcloned into p ET28a(+)plasmid which was then transformed into E.coli BL21(DE3) and induced for expression by IPTG. Expressed HBHA was purified using the Ni-NTA His-Bind Resin kit, with a concentration of 3.6 μg/μl. SDS-PAGE showed an apparent molecular weight of HBHA of approximately 28 ku. polyclonal antibodies against HBHA was prepared by immunizing the New Zealand white rabbit with purified HBHA mixed with freund adjuvant. and could be recogniozed by whole cell protein from M.bovis, in subsequent western-bloting, indicating the antigenicity of the expressed HBHA. The adhesive function of HBHA was then confirmed by co-incubation with A549 cells and observation through fluorescence microscopy.2 Construction of recombinant HBHA-mc2155 and identification of its adhesion function.The recombinant plasmid p MV361-hbha was constructed and transformed into Mycobacterium smegmatis mc2155. western bloting showed a single band at the molecular weight of about 28 ku, which indicated that HBHA was successfully expressed in mc2155. Then surface distribution of HBHA in HBHA-mc2155 cells was confirmed by fluorescence microscopy where m Ab against His tag was used as the first antibody and Goat anti mouse Ig G-FITC as the second antibody. The adhesion of recombinant HBHA-mc2155 on the surface of A549 cells was determined by confocal lasser scanning microscopy where HBHA-mc2155 and A549 cells were co-incubated, and visualized using m Ab against his tag as the first antibody, Goat anti mouse Ig G-FITC as the second antibody, when the adherence can be inhibited by polyclonal antibody against HBHA. Quantitative analysis of adhesion level of HBHA-mc2155 and mc2155 to A549 cells was determined by flow cytometry in parallel, using rabbit antibody against mc2155 as the first antibody and Goat anti rabbit Ig G-FITC as second. 49% HBHA-mc2155 adhering to A549 cells, showed a significantly higher adhesive capability of HBHA-mc2155 when compared with mc2155. And the adherence could be inhibited by HBHA protein.3. Pathogenicity of HBHA-mc2155.BALB/c mice were infected by with HBHA-mc2155 and mc2155, through intraperitoneal injection. the lung was collected to do paraffin section and stained by H. E. Microscopic examination showed that HBHA-mc2155 caused widening of the alveolar septum and infiltration of lymphocytes to the alveolar space.