基于抗原表位分析的P-糖蛋白表达研究

Expression of P-glycoprotein based on antigenic epitopes analyses

目的:对P-糖蛋白抗原表位进行分析和表达研究。方法:联合运用多种方法对P-糖蛋白的二级结构和表面特性进行分析,运用化学和酶法相结合的技术合成P-糖蛋白基因,再克隆入表达载体pGEX-2T进行表达、鉴定及纯化。结果:位于P-糖蛋白胞外段的抗原表位位点仅见于氨基酸残基82aa～115aa(Ⅰ)、741aa～760aa(Ⅳ)及800aa～816aa(Ⅴ)。SDS-PAGE分析,表达出约29 kD大小的蛋白。结论:该基因片段的高效表达为进一步制备其抗体或筛选其特异性结合肽,进行靶向治疗等工作奠定了基础。

Objective：To analyze antigenic epitopes and express P-glycoprotein. Methods： The target gene was synthesized by the combined use of chemical synthesis and Klenow polymerase extension.The synthesized gene was cloned into expression vector pGEX-2T and expressed, identified and purified it. Results： Antigenic epitopes in extracellular fragment of P-glycoprotein were identified to amino acid residues 82aa-115aa（ Ⅰ ） ,741aa-760aa（Ⅳ ）, and 800aa-816aa（ Ⅴ ）. SDS-PAGE analysis indicated that the expressed protein was about 29 kD.Conelusion：High level expression of the target gene fragment was established for preparation of its antibody and biospanning its specific peptide using random phage library, which establishes basis for the targeting treatment of MDR.