人ATP11A和ATP11B克隆、表达及鉴定  

Cloning,expression and identification of human ATP11A and ATP11B

作　　者：韩甜甜[1] 曾靖[1] 尹婕[1] 李晓莹[1] 金滢[1] 李艳[1] 潘凌亚[1] HAN Tian-tian;ZENG Jing;YIN Jie;LI Xiao-ying;JIN Ying;LI Yan;PAN Ling-ya(Department of Obstetrics and Gynecology, National Clinical Research Center for Obstetrics & Gynecologic Disease,Peking Union Medical College Hospital, CAMS & PUMC, Beijing 100730, China)

机构地区：[1]中国医学科学院北京协和医学院北京协和医院妇产科国家妇产疾病临床研究中心,北京100730

出　　处：《基础医学与临床》2022年第5期732-739,共8页Basic and Clinical Medicine

基　　金：国家自然科学基金(81572564)。

摘　　要：目的构建人P4型磷脂转移酶11A或11B(ATP11A或ATP11B)表达载体,并检测其上调ATP11A或ATP11B的效果。方法根据ATP11A或ATP11B序列设计引物,克隆到克隆载体(EZ-T^(TM)Cloning Vector)上,通过酶切鉴定,并经测序验证。分别将ATP11A和ATP11B序列酶切、定向连接到表达载体pLVX-IRES-mCherry、pLVX-IRES-RFP上。然后利用脂质体将重组带有免疫荧光蛋白的表达质粒转染至HEK-293T,最后通过测序、荧光显微镜检、流式细胞测量术分析、免疫印迹对重组细胞株进行表达验证。结果通过电泳及酶切鉴定证实了ATP11A、ATP11B表达载体的成功构建,荧光显微镜定性观察质粒转染效率,流式细胞测量术检测转染效率分别为38.9%、41.9%,免疫印迹法只检测到转染表达质粒的细胞有标签蛋白而对照组均无蛋白表达。结论成功构建了人ATP11A、ATP11B的表达载体,并可有效上调其表达水平,为将来应用ATP11A或ATP11B进行机制研究奠定了基础。Objective To construct the expression vectors carrying human TypeⅣ-P-type phospholipids transferases 11A or 11B(ATP11A or ATP11B),and to detect the up-regulation effect on ATP11A or ATP11B.MethodsThe primers were designed according to ATP11A or ATP11B sequence and cloned into EZ-T^(TM) Cloning Vector.The primers were identified by enzyme digestion and verified by sequencing.ATP11A and ATP11B sequences were digested and directed to expression vectors pLVX-IRES-mCherry and pLVX-IRES-RFP,respectively.Then the recombinant expression plasmids with immunofluorescence protein were transfected into HEK-293T using liposomes.Finally,the expression of the recombinant cell strains was verified by sequencing,fluorescence microscopy,flow cytometry and Western blot.Results The construction of ATP11A and ATP11B cloning vectors was confirmed by gel electrophoresis and was analyzed by endonuclease.The efficiency of transfection was observed qualitatively under microscope,detected quantitatively by flow cytometry as 38.9%,41.9%respectively,and the tagged proteins were detected by Western blot to present only in the positive groups.Conclusions The expression vectors of human ATP11A and ATP11B are successfully constructed and their expression levels could be effec-tively upregulated,which lay a foundation for future mechanism studies involved to ATP11A or ATP11B.

关 键 词：ATP11A ATP11B 过表达 分子克隆 

分 类 号：Q291[生物学—细胞生物学]