带有Flag标签的ZNF580真核表达载体的构建及扩增  

Construction and amplification of ZNF580 eukaryotic expression vector tagged with Flag

作　　者：罗玉玉[1] 赵瑛[1] 孔麟麟[1] 张文成[1] 

机构地区：[1]中国人民武装警察部队后勤学院生理学与病理生理学教研室,天津300162

出　　处：《中国老年学杂志》2013年第9期2062-2064,共3页Chinese Journal of Gerontology

基　　金：国家自然科学基金面上项目(81170106);天津市自然科学基金(10JCYBJC26500);武警后勤学院面上项目(WYM2010-5)

摘　　要：目的构建并扩增带有Flag标签的ZNF580真核表达载体,用于免疫共沉淀实验。方法 pGB-ZNF580质粒进行SfiI酶切,琼脂糖电泳,切胶并纯化回收ZNF580片段,与同样酶切的真核表达载体pCDEF-Flag连接构建重组质粒pCDEF-Flag-ZNF580。重组质粒转化细菌感受态,铺于氨苄抗性LB平板,37℃培养箱过夜,挑取转化了重组质粒的单克隆接种于液体LB培养基中摇床培养4～8 h。从大肠杆菌中提取质粒酶切鉴定及送Takara公司测序鉴定。结果成功构建重组质粒pCDEF-Flag-ZNF580并转化细菌感受态,于大肠杆菌中扩增得到足够用于细胞转染和免疫共沉淀实验的重组质粒,Takara测序结果显示重组质粒序列完全正确。结论成功构建和扩增了重组质粒pCDEF-Flag-ZNF580,为后续进行真核细胞的转染及免疫共沉淀实验奠定了基础。Objective To construct and amplify the ZNF580 eukaryotic expression vector tagged with Flag and used to the co-immunoprecipitation experiment. Methods The plasmids of pGB-ZNF580 and pCDEF-Flag were digested by the SfiI restriction enzyme. The aim fragments were separated by agarose electrophoresis. Then the aim fragments were purified and ZNF580 fragments were ligated into pCDEF- Flag. The recombinant plasmid pCDEF-Flag-ZNF580 was transformed into the E. coli competence. The co-transformants were plated on the LB selective media and incubated at 37℃ overnight. The monoclone＇was inoculated into the fluid LB media and incubated 4 - 8 h. The recombinant plasmids were extracted and verified by restriction enzyme digestion and sequencing. Results The recombinant plasmids were constructed successfully and transformed into the E. coli competence. The sequences of the recombinant vector were verified by sequencing. conclusions The recombinant plasmids are constructed and amplified successfully. The recombinant plasmids could be used to the transfection and co-immunoprecipitation assay, which provide basis for additional research to further investigate the functions of ZNF580.

关 键 词：ZNF580 转录因子 真核表达载体 

分 类 号：R392[医药卫生—免疫学]