Construction And Expression Of The Plasmids Expressing HBV Gene(S、X) And Function Of The Genes

Objective To construct the eukaryotic expression plasmids of the sgene and x gene of HBV, and to capture and confirm the proteins interactedwith HBx.Methods The gene fragments of Large Surface antigen、MiddleSurface antigen、Small Surface antigen and X of HBV were amplified byPCR. Then the target DNA fragments were inserted into eukaryoticexpression vector pCMV-tag2B separately to construct the recombinantplasmids pCMV-tag2B-LS、 pCMV-tag2B-MS、 pCMV-tag2B-SS andpCMV-tag2B-X; then the recombinant plasmids were sequenced. Aftersequencing and confirmation, the target plasmids pCMV-tag2B-LS、pCMV-tag2B-MS、pCMV-tag2B-SS and pCMV-tag2B-X were transfectedinto293FT cells respectively. Western blot verificated the gene fragmentswere expressed effectively. And FLAG affinity chromatographic columnwas used to separate HBX complex that was then tagged by iTRAQ afterthis. The candidate proteins were identified by tandem mass spectrometrythat also used for the analysis of the control group. Experiment was carriedout three times，and only the proteins separated from the HBx complexwere chosen as candidate protein for further research on its function. Co-Immunoprecipitation and immuno-colocalization were applied toconfirm the interaction between the candidate protein G6PD and HBx.Result1. Recombinant plasmids pCMV-tag2B-LS、 pCMV-tag2B-MS、pCMV-tag2B-SS and pCMV-tag2B-X were separately digested into twofragments with restrictive enzyme respectively. One is the same fragmentmatched the length of pCMV-tag2B vector, the other four were respectivelymatched the length of the target gene fragments(LS、MS、SS and X). AndDNA sequencing indentified the recombinant plasmids constructedincluding the complete reading frame of S gene and that of X generespectively.2. Western blot confirmed four strong reaction bands at43KDa、34KDa、24KDa and17KDa corresponding to the recombinant plasmidspCMV-tag2B-LS、 pCMV-tag2B-MS、 pCMV-tag2B-SS andpCMV-tag2B-X respectively, and no reaction bonds for the blank control.3. HBx complex was separated using FLAG affinity chromatographiccolumn; and58candidate proteins including G6PD were identified usingtandem mass spectrometry.4. The interaction between the candidate protein G6PD and HBx wereconfirmed using Co-Immunoprecipitation and immuno-colocalization.Conclusion successfully the eukaryotic expression plasmids of the sgene and x gene of HBV were constructed and expressed, and the HBVcomplex was separated and the proteins related to HBX protein wereidentified. The interaction between G6PD as a candidate proein and HBxwas confirmed by tandem mass spectrometry. All of this could lay thefoundation for the further research on the function of HBS、HBx.