摘要
目的 以乙型肝炎病毒e抗原 (HBeAg)全基因为外源片段 ,构建酵母双杂交体系中结合域载体 ,研究其在酵母细胞中的表达 ,排除自身激活作用及毒性作用 ,验证其适用性。方法 根据报道序列设计、合成HBeAg全基因巢式引物 ,取患者血清 ,通过PCR方法分离HBeAg全基因片段 ,克隆入T载体 ,酶切鉴定及序列测定后 ,克隆入酵母双杂交体系结合域载体pGBKT7,构建pGBKT7 eAg载体。再次酶切鉴定阳性克隆后转入酵母 ,验证毒性作用 ;通过Western blot实验证实外源基因在酵母中的表达 ;并进一步验证自身激活作用。结果 由血清中分离的及 2次酶切鉴定的片段大小分别为 6 5 7bp和 6 39bp ,与预期大小一致 ;序列测定与报道的HBV分离株核酸及氨基酸同源性分别为96 %和 10 0 % ;转人酵母后无毒性及自身激活作用 ;Western blot实验出现阳性与预期大小 (2 4 .3× 10 3)一致的条带。结论 pGBKT7 eAg载体在酵母细胞中表达出融合蛋白 ,进一步验证无自身激活及毒性作用 ,此载体构建成功 ,适用于酵母双杂交体系。
Objective Using hepatitis B virus e antigen (HBeAg) gene to construct the DNA-binding domain vector,which can express HBeAg in yeast cell,and can be used in yeast double hybrid as 'bait plasmid' to look for the gene from the cDNA library,which expresses the protein that can interact with HBeAg. Methods PCR was performed to amplify the HBeAg gene from a sera of hepatitis B patient. The product of the amplification was inserted into T-vector and was verified by sequencing. Then it was inserted into the 'bait' plasmid pGBKT7 after the digestion with the restricted endonuclease of EcoR I and Sal I. The plasmid was transformed into the yeast cell. PCR was used to verify whether the plasmid was transformed into yeast. The HBeAg protein expressed in the cell was confirmed by Western blot. Using nutrition selection assay to verify the constructed plasmid alone could not activate the reporter gene in the yeast cell.Results Sequenced and digested by two endonuc1eases,the recombined vectors pGBKT7-eAg produced anticipated fragment. PCR verified that there was HBeAg fragment in the yeast. Having assayed by Western blotting,it was shown that the yeast cell transformed with pGBKT7-eAg vector had positive signal which could not be seen in the control. Tested by the nutrition selection assay,the recombined vectors pGBKT7-eAg could not activate LacZ reporter gene in the yeast.Conclusion DNA-binding domain plasmid was successfully constructed and could express HBeAg proteins in the yeast cell but could not activate transcription of LacZ reporter gene alone. The recombined plasmid can be used in yeast double hybrid.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2004年第2期158-161,共4页
Chinese Journal of Experimental and Clinical Virology
基金
国家自然科学基金资助 (3 0 10 0 170 )
