Cloning, Expression And Purification X Protein Of The Genotypes B And C Of Hepatitis B Virus In The Prokaryotic Expression System

Objective: To obtain the X antigen from the Genotypes B and C of Hepatitis B Virus by genetic engineering and protein technology. To establish the base of making further study the X protein'function in the diseases which are caused by Hepatitis B Virus.Method:①Gene cloning: The X gene from the conservative genotypes B and C of HBV was amplified by using PCR from the eukaryotic expression vectors , pcDNA3.1-Xb and pcDNA3.1-Xc,which is constructed in our lab. After digested with EcoRI and XhoI,the gene fragments and expression vector pET-42a were ligated respectively.The recombinant vector were transformed into BL-21 competent cell. The correct recon were identifyied by antibiotics, PCR,enzyme digestion and sequencing.②Protein expression and purification: Recombination protein were detected by GST antibody with SDS-PAGE and Western Breeze chemiluminescent after induced by IPTG. The time and IPTG density of the induction were Optimized to obtain the max protein. To get the dissoluble protein ,it is tried by inducing the fusion protein at low temperature.Inclusion body were dissolved by urea.and affinity purified by GST-Tag and Ni-NTA His.Bind . The density of the recombination protein was determined with Bradford.Results:①Homologies of the nucleotide and putative amino acid sequence of the X gene were perfectly compared to the reported sequences respectively.The positive recombinant plasmid were improved correctly by PCR,digest and sequencing. ②The X fusion protein of genotypes B and C could induced by IPTG and the expressed products could be recognized by GST antibody. The maximum expressed quantity were as high as 40% above the total bacterium protein when induced with 0.5/mmol /L IPTG for 2 hours. It is difficult to induce the dissoluble protein at 18℃,25℃,30℃.the Inclusion body was dissolved with 8mol/L urea and then was refolded by using the Protein Refolded Kit.We can't get the recombination protein after eluting the GST bind in the end. However ,using Ni-NTA His.Bind, the fusion protein was obteined by using the denatured protein straightly. The purity quotient of recombination protein reachs up to more than 95% after affinity purification.Conclusion:The prokaryotic expression system with'X genes of the genotypes B and C of HBV were successfully constructed in our study.The recombination protein could be induced efficiently.We can get high purity X fusion protein after using Ni-NTA bind. the X fusion proteins will be established the base of making further study the X protein'function in the diseases which are caused by Hepatitis B Virus.