Cloning And Expression Of Hepatitis C Virus Core Protein Binding Protein 12, And The Study Of Its Function

Background and Objective Hepatitis C caused by the infection of Hepatitis C virus (HCV) is one of the main causes of chronic Hepatitis and the cause of hepatic cancer; however, there are lack of efficient therapies for HCV infection nowadays. Study the pathogenetic mechanism of HCV is of great importance. On the base of having found the hepatocyte protein interacting with HCV core protein, and one new gene with unknown functions named HCV Core protein binding protein 12 (HCBP12), the purpose of the study is to clarify biological function of HCBP12 in the course of HCC through the following aspects, such as subcellular orientation, prokaryotic expression, antibody preparation and screening of binding proteins in hepatocytes.Methods (a) Construction of recombined green fluorescent protein expressive vector pEGFP-C1- HCBP12. HepG2 cells were transfected, and observed by fluorescent inverted microscope after 24hr. (b) The prokaryotic expressive vector pET-32a(+)-HCBP12 was constructed and transformed into the competent BL21 E. coli. The HCBP12 protein was induced with IPTG and analyzed using SDS-PAGE and identified using Western blot. The expressed product was purified by Ni+ affinity column chromatography. The purified pET-32a(+)-HCBP12 fusion protein was used to immunize New Zealand rabbits to produce polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA. (c) The yeast expressive vector pGBKT7- HCBP12 was constructed and transformed into AH109 yeast strains. The yeast protein was isolated and detected by Western blotting analysis. Yeast two-hybrid was performed by mating AH109 with Y187 containing liver cDNA library plasmid to screen proteins interacting with HCBP12.Results (a) HCBP12 can be subcellularly located in cytoplasm by its visible green fluorescent signal. (b) The HCBP12 fusion protein was highly expressed in the prokaryote expressing system. The protein production mainly was in inclusion body through SDS-PAGE analysis. The purified protein and polyclonal antibody were obtained successfully. ELISA manifested the titer of polyclonal antibody >1:512,000. The high specificity was testified using Western blot. (c) The HCBP12 fusion protein was expressed successfully in the yeast strains by Western blotting analysis. There were twelve different hepatocyte proteins interacting with HCBP12 by Yeast two-hybrid, containing human sapien apolipoprotein C-I/B, human sapiens mitochondrion, human sapiens metallothionein 2A, human sapiens beta-2-microglobin,human sapiens carboxylesterase, and human sapiens serpin peptidase inhibitor, with known function and one with unknown function. The new gene was submitted to GenBank database and named HCBP12BPA.Conclusions Our work proved that HCBP12 subcellularly locates in cytoplasm. HCBP12 can combine with apolipoprotein, metallothionein 2A, beta-2-microglobin and carboxylesterase.These proteins are very important in the course of hepatocellular proliferation, apoptosis and metabolism. The HCBP12 fusion protein was induced and expressed successfully by E. coli prokaryote expressing system. The expressed protein was purified by affinity column chromatography and highly specific and potent polyclonal antibody of HCBP12 was prepared. The above results provided an valuable information for the further study on the meaning of HCBP12 in the course of HCC by other methods such as immune histochemistry, mammal two-hybrid, cellular proliferation and signal transduction.