Construction Of The Fusion Protein Ag85B-Hsp16.3 Of Mycobacterium Tuberculosis And Its Effects On Human Hepatoma Carcinoma Cell HepG-2

Liver cancer is one of the most common and lethal malignant tumors. Our country has a high risk of liver cancer, so it is significantly important for us to focus on the diagnosis and treatment of liver cancer. At present, partial resection together with radiation therapy and chemotherapy are predominantly adopted to treat liver cancer in the clinics. In 1976, Morales A et al. reported the treatment effectiveness of Bacillus Calmette Guerin(BCG) on superficial bladder tumors the first time, which was widely accepted, and then immunotherapy against tumors became a hot spot.The concrete mechanisms of immunotherapy against tumors are still undefined. Zhang Zizhen et al. used different concentrations of Mycobecterium Tuberculosis(MTB) supernatant to treat human hepatoma carcinoma cell HepG-2 and found that moderate concentrations of MTB supernatant could inhibit the proliferation of HepG-2 cells. However, MTB supernatant is consisted of many components, including MTB secreting proteins such as Ag85B, ESAT6, Hsp16.3, CFP10 and so on. Which component of MTB supernatant inhibiting the proliferation of HepG-2 still needs to be lucubrated.Our laboratory has successfully constructed many MTB secreting proteins and fusion proteins, such as Hsp65, Hsp16.3, Ag85B-ESAT6, HSP65-IL-2, ESAT6-CFP10 and so on. Ag85B is an important protective antigen in both MTB and BCG supernatant, which can induce significant Th1 immune response in vivo. The level of Hsp16.3 increases the most when MTB is at dormancy, so Hsp16.3 is closely related to MTB latent infection and dormancy. Thus, we choose these two proteins to construct a fusion protein, followed by detection of effects of the fusion protein on HepG-2 cells, to investigate whether it can inhibit the proliferation of HepG-2 or not. AIM:To construct the fusion protein Ag85B-ESAT6 of MTB and investigate the effects of Ag85B-Hsp16.3, Ag85B-ESAT6 and Hsp16.3 on HepG-2 in vitro.METHODS AND RESULTS:1. Construction, expression and purification of the fusion protein Ag85B-Hsp16.3The Hsp16.3 gene was amplified by PCR from the plasmid pProEX HTb-Hsp16.3 preserved in our lab, and a flexible chain with 48bp was linked between the Ag85B gene and the Hsp16.3 gene in order to ensure the correct folding of the protein. The Hsp16.3 gene was then cloned into vector pMD18-T, followed by analysis with DNA sequence. The sequence of the Hsp16.3 gene was in accordance with that of the GenBank, and the Hsp16.3 gene took the place of the ESAT6 gene in the recombinant plasmid pProEX HTa-Ag85B- ESAT6 that was preserved in our lab. After analysis with enzymatic digestion, the positive recombinant plasmid was named pProEX HTa-Ag85B-Hsp16.3 and the fusion protein was induced by IPTG in E.coli DH5α. Following analysis of SDS-PAGE in which we got the aimed protein, analysis of Western blot was done to make sure that the fusion protein could response specifically to Ag85B polyclonal antibody, Hsp16.3 monoclonal antibody and active TB patient's serum, respectively. The fusion protein was purified by Ni-NTA purification system under denaturing condition, and it mainly existed in inclusion body.2. Detection of effects of Ag85B-Hsp16.3, Ag85B-ESAT6 andHsp16.3 on HepG-2 cells by MTT test assayThe plasmids pProEX HTa-Ag85B-Hsp16.3, pProEX HTa-Ag85B-ESAT6 and pProEX HTb-Hsp16.3 were transferred into E.coli DH5αand induced by IPTG. Following analysis of SDS-PAGE, the three proteins were purified by Ni-NTA purification system under denaturing condition. After renaturation and filtration, the three proteins were respectively added into HepG-2 cells with different concentrations and different action time. The MTT test results showed that moderate concentrations of the three proteins could all inhibit the proliferation of HepG-2 cells, dependent on the concentration as well as the action time. But there were no significant differences between the effects of different proteins.CONCLUSIONS1. The fusion protein Ag85B-Hsp16.3 was successfully constructed and could response to Ag85B polyclonal antibody, Hsp16.3 monoclonal antibody and active TB patient's serum respectively. In E.coli DH5α, the protein was expressed efficiently. 2. The three proteins Ag85B-Hsp16.3, Ag85B-ESAT6 and Hsp16.3 were successfully induced by IPTG and purified. After renaturation, the three proteins could all inhibit the proliferation of HepG-2 cells, dependent on the concentration and the action time. No significant differences were found between different proteins.