Construction And Preliminary Functional Analysis Of Over-expression Vector And RNAi Expression Vector Of HCV Core Protein Binding Protein 6 (HCBP6)

HCV is one of main cause of the chronic hepatitis , hepatic cirrhosis and carcinoma, almost 250,000～350,000 die from the HCV and related disease . So,it is no denied that HCV is a serious problem of society and public health, nevertheless , we have no more effective therapeutic method apart from the united of interferon and ribavirin, what is worse is that we have no vaccine for HCV and it is useless to the patient of decompensated period. Further reveal HCV pathogenesis of current research is the main difficulties ,Further explore HCV pathogenic molecular biology mechanism also became one of the hot topics in current researches.We get a kind of protein called HCV core protein binding protein 6 (HCBP6) which can combined with hepatitis c virus core protein, is screened by Our laboratory experiments in prophase yeast double hybrid technology application from liver cells in cDNA library . we find that HCBP6 can up-regulation and down-regulation the expression of some gene, and this differential expression be correlated with the signal transduction, hyperplasia, differentiation and the growth control of the cell. So it is necessary for us to have a further research of the HCBP6.Early days we studied the regulation mechanism of HCV core protein binding protein 6 (HCBP6). We forecasted HCBP6 gene promoter on transcription factor binding sites by relevant bioinformatics technology. I checked the active region of promoter by stepwise deletion. I also use EMSA ( Electrophoretic Mobility Shift Assay) to check the active area and the area of combination of transcription factor. Now we have constructed Over-expression Vector and RNAi Expression Vector of HCV core protein binding protein 6 ,Through selecting and cell scratch test, we get two high suppressed efficiency RNAi carriers,and obtain preliminary understanding on the presumable function of HCBP6.ObjectiveResearch the effect about the protein HCBP6 on biological behavior of HepG2 cells,establish foundation for further studies about the role of HCBP6 in HCC. Methods1. Constructing the Over-expression Vector: labeled the vector with c-myc and His tags, and prepare for the later experiment. 2. Constructing the RNAi Vector: judge the transfection efficiency and extract total RNA, get the cDNA through reverse transcription. Then we select two high suppressed efficiency RNAi carriers for the later experiment.3.Through preliminary functional experiment , research the effect about the protein HCBP6 on biological behavior of HepG2 cells,Study the feasible function of the geneResults1.We have constructed the Over-expression Vector: pcDNA3.1(-)-myc/his-HCBP62.We have constructed the RNAi vector; through sequencing we prove the vector is right. The express activity of the experimental group is 3/5 of the control group. We obtain the two higher suppressed vectors.3.We find that HCBP6 can promote the migration of HepG2 cell. Preliminarily induce that HCBP6 can promote the tumor metastasis.ConclusionsIn this research we come to a conclusion that HCBP6 can positively regulate the biological behavior-migration in hepatocellular liver carcinoma cell line HepG2 cell.