Study On Binding Site Of HBV X Protein Interacting With Cytochrome C Oxidase Ⅲ

AIM: To identify the binding site of hepatitis B virus X protein (HBx) interacting with cytochrome c oxidase III (COXIII) by yeast two-hybrid system.METHODS: Two mutants of HBV X(X1 1-72aa;X2 1-117aa),obtained by normal polymerase chain reaction(PCR), were inserted into multiple cloning site (MCS) of yeast expression plasmid pAS2-1,which contains GAL4-BD.Then pAS2-1/(X mutants) were transformed into the yeast AH109 by lithium acetate-method,pAS2-1/X as control. Expression of HBV X and X mutants-BD fusion protein in yeast cells were identified by Western Blot. Filter assay was used to detect the auto-activation function of pAS2-1/(X mutants) in AH109. pAS2-1/X and pAS2-1/(X mutants) were transformed into AH109,while pACT2/COXIII was transformed into Y187. The yeast cells cotransformed with pAS2-1/(X mutants) and pACT2/COXIII were cultured in SC/-trp-leu. The second screening was performed withβ–gal activity detection.RESULTS: The constructed pAS2-1/(X mutants) were verified by digestion of restriction endonuclease, PCR and auto-sequencing. pAS2-1/(X mutants) correctly expressed BD-X mutants fusion protein in yeast AH109 by PCR and Western Blot. Filter assay was used to exclude the auto-activation function of pAS2-1/(X mutants) in AH109. The yeast cells cotransformed with pAS2-1/(X mutants) and pACT2/COXIII grew in selective SC/-trp-leu. But none of the clones passed through theβ–gal activity detection.CONCLUSION: Bait plasmid pAS2-1/(X mutants) were successfully constructed and pAS2-1/(X mutants) correctly expressed BD-X mutants fusion protein in yeast AH109. pAS2-1/(X mutants) didn't have auto-activation function in filter assay in AH109.pAS2-1/ X1 and pAS2-1/ X2 didn't include binding site of HBV X protein interacting with COXIII so the binding site most probably in HBx 118-154 amino acids.