人端粒重复序列结合因子1^(33-277)在大肠杆菌中的克隆及高效表达  

Cloning and High Level Expression of Telomeric Repeat Binging Factor1^(33-277) in Escherichia Coli

作　　者：黄河[1] 陈巧芳[1] 林茂芳[1] 

机构地区：[1]浙江大学医学院附属第一医院,浙江杭州310003

出　　处：《浙江大学学报（医学版）》2000年第5期193-195,202,共4页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金资助项目(基金编号39870339)

摘　　要：目的 :建立能高效表达人端粒重复序列结合因子 1第 33- 2 77位 (TRF1 33- 2 77)氨基酸的菌株并制备多抗。方法 :设计一对引物 ,分别带有 Kpn 和 Eco RI位点 ,从已经克隆了 TRF1 c DNA全长的重组质粒 p CR -TOPO-TRF1扩增 TRF1 33- 2 77c DNA片段 ,克隆入 PGEM-T质粒 ,测序和酶切图谱鉴定 ,Eco RI酶切后亚克隆入 p GEX-3X的 Eco RI位点之间 ,筛选正向插入重组子。转化大肠杆菌 DH5 a,筛选高表达菌株。结果 :得到高效表达融合蛋白的菌株 DH5 a-TRF1 33- 2 77。其融合蛋白经western blot证实具 TRF1抗原性。结论 :所建立的菌株为 h TRF1单克隆和多克隆抗体制备提供蛋白来源。Objective:To obtain a plasmid transfected E.coli that can express human telomeric repeat binding factor (TRF)1 33 277 at high level.Methods:Two primers were designed with Kpn I and EcoRI sites respectively,TRF1 33 277 cDNA fragment was amplified from recombinent plasmid pCRⅡTOPO TRF1,which contains full length of TRF1 cDNA.The fragment was cloned into plasmid pGEMT,confirmed by sequencing and restriction endonuclease map analysis,then subcloned into E.coli DH5a.The fusion protein was tested by SDS PAGE and confirmed by western blot.Results:E.coli DH5a TRF1 33 277 expressing high level of TRF1 33 277 was obtained,and purified GST TRF1 33 277 was also obtained.Conclusion:E.coli DH5a TRF1 33 277 can be the protein source for production of polyclonal or monoclonal anti hTRF1 antibodies.

关 键 词：端粒 端粒重复序列结合因子 基因 细菌 重组融合蛋白质类 

分 类 号：R378[医药卫生—病原生物学]