摘要
AIM: To seek the X associated protein (XAP) with theconstructed bait vector pAS2-1X from normal human livercDNA library.METHODS: The X region of the HBV gene was amplied byPCR and cloned into the eukaryotic expression vector pAS2-l.The reconstituted plasmid pAS2-1X was transformed intothe yeast cells and the expression of X protein (pX) wasconfirmed by Western blot analysis. Yeast cells werecotransformed with pAS2-1X and the normal human livercDNA library and were grown in selective SC/-trp-leu-his-ade medium, the second screen was performed with theLacZ report gene. Furthermore, segregation analysis andmating experiment were performed to eliminate the falsepositive and the true positive clones were selected for PCRand sequencing.RESULTS: Reconstituted plasmid pAS2-1X including theanticipated fragment of X gene was proved by auto-sequencing assay. Western blot analysis showed thatreconstituted plasmid pAS2-1X expressed BD: X fusionprotein in yeast cells. Of 5 × 106 transformed coloniesscreened, 65 grew in the selective SC/-trp-leu-his-ademedium, 5 scored positive for β-gal activity, and only 2remaining clones passed through the segregation analysisand mating experiment. Sequence analysis identified thattwo clones contained similar cDNA fragment: GAACFFGCG.CONCLUSION: The short peptide (glutacid-leucine-alanine)is a possible required site for XAP binding to pX. Normalhuman liver cDNA library has difficulties in expressing theintegrated XAP on yeast cells.
AIM:To seek the X associated protein(XAP)with the constructed bait vector pAS2-1X from normal human liver cDNA library. METHODS:The X region of the HBV gene was amplied by PCR and cloned into the eukaryotic expression vector pAS2- 1.The reconstituted plasmid pAS2-1X was transformed into the yeast cells and the expression of X protein(pX)was confirmed by Western blot analysis.Yeast cells were cotransformed with pAS2-1X and the normal human liver cDNA library and were grown in selective SC/-trp-leu-his- ade medium,the second screen was performed with the LacZ report gene.Furthermore,segregation analysis and mating experiment were performed to eliminate the false positive and the true positive clones were selected for PCR and sequencing. RESULTS:Reconstituted plasmid pAS2-1X including the anticipated fragment of X gene was proved by auto- sequencing assay.Western blot analysis showed that reconstituted plasmid pAS2-1X expressed BD:X fusion protein in yeast cells.Of 5×10~6 transformed colonies screened,65 grew in the selective SC/-trp-leu-his-ade medium,5 scored positive for β-gal activity,and only 2 remaining clones passed through the segregation analysis and mating experiment.Sequence analysis identified that two clones contained similar cDNA fragment:GAACTTGCG. CONCLUSION:The short peptide(glutacid-leucine-alanine) is a possible required site for XAP binding to pX.Normal human liver cDNA library has difficulties in expressing the integrated XAP on yeast cells.
