人血清白蛋白与PTH(1-34)融合蛋白在毕赤酵母中的分泌表达及鉴定  

Construction,expression and characterization of recombinant fusion protein HSA-PTH(1-34) in pichia pastoris

作　　者：陈静[1] 孙红颖[1] 杨颖[1] 王学芬[1] 陈枢青[1] 

机构地区：[1]浙江大学药学院生化药学研究室,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2008年第2期126-133,共8页Journal of Zhejiang University(Medical Sciences)

基　　金：浙江省科技厅科研基金资助项目(2006C33002);浙江省卫生厅科研基金资助项目(2005A070);浙江省教育厅科研基金资助项目(20061327)

摘　　要：目的:在毕赤酵母中高效分泌表达人血清白蛋白与PTH(1-34)融合蛋白。方法:利用PCR技术获得HSA和PTH(1-34)基因,以柔性连接肽相连插入到表达载体pPIC9,重组质粒线性化后,用化学法转化毕赤酵母GS115,经组氨酸缺陷型培养板和PCR鉴定筛选得到转化子。在启动子AOX1和α交配因子信号肽的作用下,分泌表达融合蛋白HSA-PTH(1-34)。PCR法和电泳鉴定表达验证阳性转化子,并观测不同时间点的表达量。Western blot验证其免疫活性,在兔肾皮质细胞膜中测定腺苷酸环化酶的激活产生cAMP的量来检测融合蛋白的生物学活性。结果:PCR鉴定重组转化子验证了融合基因的成功整合,用甲醇诱导表达,蛋白电泳分析表明融合基因得到高效表达,HSA-PTH(1-34)同时具有HSA和PTH(1-34)的抗原性,且能激活兔肾皮质细胞中的腺苷酸环化酶产生cAMP,但活性低于PTH(1-34)。结论:在毕赤酵母中成功表达了具有生物学活性的人血清白蛋白与PTH(1-34)融合蛋白。Objective： To obtain recombinant fusion protein HSA （human serum albumin） -PTH（1-34） in pichia pastoris. Methods： HSA and PTH（1-34） cDNA were obtained with PCR and the DNA segments were cloned into vector pPIC9 with linker. The linearized plasmids were transformed GSl15 competent cells treated with LiCl,and rout＋ transformants were screened on MD plate. With AOX promoter and α-MF signal sequences leading, fusion protein was expressed in GSl15. PCR and SDS-PAGE were employed to confirm the integration and expression of HSA -PTH （1-34）. The fusion protein was identified by Western blotting and classical adenylate cyclase assay. Results. The PCR results showed that the gene of HSA-PTH（1-34） was integrated into GSl15 genome. Western bolt approved the existence of two domains of HSA and PTH（1-34）. The bioactivity assay in rabbit cortical membranes indicated that HSA-PTH（1-34） activated adenylate cyclase,but the activity was lower than that of the synthetic PTH（1-34）. Conclusion; Active fusion protein HSA-PTH（1-34） is successfully expressed in pichia pastoris.

关 键 词：毕赤酵母 甲状旁腺激素肽(1—34) 血清白蛋白 质粒 重组 遗传 人甲状旁腺激素(1—34) 融合蛋白 分泌表达 

分 类 号：Q78[生物学—分子生物学]