FLT3及FLT3-ITD突变体在HEK293T细胞的表达和纯化  

Expression and purification of FLT3 and FLT3-ITD mutated proteins in HEK293T cells

作　　者：杨凡 杜佳慧 吴洁 袁濮玉 苏倩 撖荣 邱桥成[2] 薛胜利[2] 刘松柏 孙中文[1] YANG Fan;DU Jiahui;WU Jie;YUAN Puyu;SU Qian;HAN Rong;QIU Qiaocheng;SUN Zhongwen(Suzhou Key Laboratory of Medical Biotechnology, Suzhou Vocational Health College, Suzhou 215009;Department of Hemotology, First Affiliated Hospital of Soochow University, Suzhou 215006, China)

机构地区：[1]苏州卫生职业技术学院苏州检验医学生物技术重点实验室,江苏苏州215009 [2]苏州大学附属第一医院血液科,江苏苏州215006

出　　处：《细胞与分子免疫学杂志》2018年第3期242-246,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81470296,31701198);江苏省自然科学基金(BK20170386);江苏省“科教强卫工程”青年医学人才项目(QNRC2016719,QNRC2016771);江苏省卫生计生委医学科研课题面上项目(H2017074);江苏省六大人才高峰(2016-WSN-123);江苏省教育厅大学生实践创新训练项目(201712688003Y);江苏省教育厅“青蓝工程”项目(2016);苏州卫生职业技术学院重点项目(szwzy201701)

摘　　要：目的构建fms相关酪氨酸激酶3(FLT3)及FLT3-内部串联重复序列(FLT3-ITD)突变体的真核表达载体,在HEK293T细胞中稳定表达并通过免疫沉淀技术对蛋白进行纯化。方法从正常人及FLT3-ITD阳性急性髓系白血病患者骨髓干细胞中提取RNA,反转录为cDNA,使用带有流感病毒血凝素(HA)标签序列的特异性引物,采用PCR扩增出人FLT3及FLT3-ITD突变体编码区全长并克隆到CD530A-T2A-GFP载体上。重组的FLT3及突变体FLT3-ITD表达质粒,通过脂质体polyJ et转染到HEK293T细胞进行表达。使用HA抗体结合微珠沉淀带HA标签的FLT3及FLT3-ITD突变体蛋白。然后,通过HA肽段将蛋白竞争洗脱下来,洗脱下来的蛋白经银染染色。结果 FLT3及FLT3-ITD突变体全长编码序列成功构建到CD530A-T2A-GFP载体上,Western blot法及聚丙烯酰胺凝胶银染法成功检测到FLT3及FLT3-ITD突变体蛋白的表达、纯化。结论在HEK293T细胞中成功表达并纯化FLT3及FLT3-ITD突变体蛋白。Objective To construct the eukaryotic expression vectors of human fms related tyrosine kinase 3 （FLT3） gene and FLT3-internal tandem duplication （FLT3-1TD） mutants and purify the native proteins through immunoprecipitation from HEK293T cell lysates. Methods The cDNA fragments of FLT3wt and FLT3-1TD were amplified from bone marrow cells of healthy individuals and FLT3-1TD-mutated acute myeloid leukemia （AML） patients with specific primers, and the PCR products were cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-1TD plasmids were transfected in HEK293T cells by Polyjet reagent, and the recombinant proteins were purified by immunoprecipitation and competing elution methods. Results FLT3wt and FLT3-1TD-mutated DNA sequences were successfully cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-1TD mutated proteins were successfully expressed and purified in HEK293T cells as verified by Western blotting and sliver staining. Conclusion FLT3wt and FLT3-1TD expression vectors were successfully constructed, and purified proteins were successfully obtained from HEK293T cells.

关 键 词：FMS样的酪氨酸激酶3 突变体 蛋白纯化 FMS样的酪氨酸激酶3-内部串联重复 

分 类 号：R392.11[医药卫生—免疫学] Q512[医药卫生—基础医学]