CAJ | 학술논문

目的探索细胞色素C氧化酶亚单位Ⅲ(COX Ⅲ)与乙型肝炎病毒X蛋白(HBx)的具体结合位点. 方法构建pAS2-1-X变异体重组载体,醋酸锂转化法将其转入酵母细胞,通过PCR法及测序法证实目的片段转入酵母细胞;Western blot法明确变异体蛋白在酵母细胞中能否正确表达,滤膜转印法排除自身激活作用;固体培养基交合实验及β-半乳糖苷酶活性实验检测HBx和COX Ⅲ的结合区域. 结果成功构建含HBx第1～ 72位氨基酸的变异体重组载体pAS2-1-X1和第1～ 117位氨基酸的变异体重组载体pAS2-1-X2,测序及PCR均证实片段的正确性.Western blot证实变异体蛋白在酵母细胞中可正确表达,且变异体蛋白无自身激活作用.交合实验及β-半乳糖苷酶活性检测将HBx和COX Ⅲ的结合区域定位于72 ～ 117位氨基酸. 结论通过酵母双杂合实验证实HBx和COX Ⅲ的结合区域定位于72 ～ 117位氨基酸,此结合区域的明确有望帮助进一步阐明X蛋白在体内的作用机制,并可能为慢性乙型肝炎、肝硬化及肝癌的防治提供新思路.
목적 탐색세포색소C양화매아단위Ⅲ(COX Ⅲ)여을형간염병독X단백(HBx)적구체결합위점. 방법 구건pAS2-1-X변이체중조재체,작산리전화법장기전입효모세포,통과PCR법급측서법증실목적편단전입효모세포;Western blot법명학변이체단백재효모세포중능부정학표체,려막전인법배제자신격활작용;고체배양기교합실험급β-반유당감매활성실험검측HBx화COX Ⅲ적결합구역. 결과 성공구건함HBx제1～ 72위안기산적변이체중조재체pAS2-1-X1화제1～ 117위안기산적변이체중조재체pAS2-1-X2,측서급PCR균증실편단적정학성.Western blot증실변이체단백재효모세포중가정학표체,차변이체단백무자신격활작용.교합실험급β-반유당감매활성검측장HBx화COX Ⅲ적결합구역정위우72 ～ 117위안기산. 결론 통과효모쌍잡합실험증실HBx화COX Ⅲ적결합구역정위우72 ～ 117위안기산,차결합구역적명학유망방조진일보천명X단백재체내적작용궤제,병가능위만성을형간염、간경화급간암적방치제공신사로.
Objective To identify the binding site position of the hepatitis B virus (HBV) X protein (HBx) functional interaction with the cytochrome C oxidase subunit Ⅲ (COX Ⅲ,a key regulator of mitochondrial function) by using a yeast two-hybrid system.Methods Two fragments of HBx mutants (X1 1-72aa and X2 1-117aa) were amplified by PCR and inserted into the bait plasmid pAS2-1.The resultant mutant plasmids were transfected into yeast cells using the lithium acetate-method.PCR and gene sequencing were used to confirm that the mutant fragments were expressed properly in yeast cells.Western blotting was used to verify that the mutant proteins were translated accurately in the yeast cells.Filter assay was used to exclude autoactivated mutants.Hybridization in solid medium and β-gal activity detection were used to determine the precise position of the binding site for HBx and COX Ⅲ interaction.Results The two mutant plasmids containing HBx 1-72aa and 1-117aa respectively were successfully constructed and the mutants were both properly expressed and translated in yeast cells; no autoactivated mutants were detected throughout the experimental process.The binding site of HBx and COX Ⅲ was found to be encompass the amino acids 72 through 117 of HBx.Conclusion Amino acids 72 through 117 of HBx are the key domain of the HBx functional interaction With COX Ⅲ; this domain may represent a useful target for molecular-based therapies to treat HBV-related diseases.