Confirmation Of Aldolase B As A Novel Binding Protein Of The Major S Protein Of Hepatitis B Virus And Study On The Function Of Aldolase B

Objective: TO confirm the candidate hepatocyte binding protein ALDOB interacting with the surface antigen protein of hepatitis B virus and study on the function of Aldolase B.Methods:(1) ALDOB gene was amplified by RT-PCR with the total RNA of human HepG2 cell as template.The PCR fragments were cloned into pCMV5 vector to construct recombinant eukaryotic expression plasmids.Whole S gene recombinant plasmid as a template by PCR amplification of the major S gene ,building PCMV4-Flag-SHBs expression vector. 293T cells were transfected with the recombinant plasmids. Immunofluorescence staining and Western blot were used to certificate the expressions of ALDOB-myc and major S in 293FT cells.(2)The two plasmids were co-transfected into 293FT, followed by confocal laser microscopy analysis to identify the co-localization of major S and ALDOB proteins. SHBs-flag gene expression plasmid was transfected into HepG2 cells to establish stable transfected major S protein cell line, using the co-immunoprecipitation method to verify binding of the major S protein and ALDOB protein. (3) Construction of recombinant targeting aldolase B interference plasmid siALDOB-Pu6, transfecting into HEPG2 cells to select the stability of interference plasmid transfer lines. By cell proliferation assays, Transwell experiments, apoptosis experiment, explore the role of ALDOB in HepG2 hepatoma cells by the growth, invasion and apoptosis. Results: (1) The sequence of ALDOB and major S were 100% homology with difference human gene previously registered in GenBank. The result of Immunofluorescence staining manifest: recombinate protein of ALDOB-myc protein expression in 293FT cells;Major S protein express in the cytoplasm.An interesting band about 40kD was visible in the resuult of Western blot, which was consistent with expected with expected size of recombinate protein of ALDOB-myc expressed in 293FT cells; About 26KD in the expected location of the Major S protein. (2)Co-immunoprecipitation method is successfully used for verifying of ALDOB protein as an interacting protein of surface antigen protein for hepatitis B virus.(3) Specific inhibition ALDOB expression of the HepG2 cell line, cell proliferation accelerated,invasion capacity enhanced and apoptosis rate increased than not inhibited ALDOB in HepG2 cell line.Conclusion:(1)pCMV5-ALDOB-myc and pCMV4-majorS-flag have been constructed successfully.(2)ALDOB protein is novel binding protein of the major S protein of hepatitis B virus.(3) ALDOB can inhibit HepG2 cells proliferation,invasion ability and inhibit HepG2 cell apoptosis.