pNTAP-PRAK真核表达载体的构建及其在HEK293细胞中的表达  

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

作　　者：王达安[1,2] 龚小卫[1] 刘爱华[3] 梅柱中[1] 王丹[1] 明小燕[1] 王旭[1] 魏洁[1] 邓鹏[1] 姜勇[1] 

机构地区：[1]南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室 [2]暨南大学医学院病理生理学系,广东广州510632 [3]南方医院呼吸科,广东广州510515

出　　处：《中国病理生理杂志》2010年第9期1813-1817,共5页Chinese Journal of Pathophysiology

基　　金：长江学者和创新团队发展计划资助项目(NoIRT0731);国家自然科学基金委员会-广东省人民政府自然科学联合基金重点资助项目(NoU0632004);国家自然科学基金资助项目(No30670828;No30572151;No30700291);高等学校博士学科点专项科研基金资助项目(No20069981001)

摘　　要：目的:构建pNTAP-PRAK真核表达质粒,并建立其稳定表达的HEK293细胞系。方法:将人的PRAK亚克隆至串联亲和纯化(tandem affinity purification,TAP)载体pNTAP质粒上,构建成重组质粒pNTAP-PRAK,转化该重组质粒至感受态大肠杆菌DH5α,阳性克隆进行PCR、酶切及DNA测序验证正确后,利用PolyFect脂质体介导将其转染至HEK293细胞中,再通过G418筛选建立稳定表达TAP tag-PRAK融合蛋白的HEK293细胞系;利用Western blotting和细胞免疫荧光标记法检测融合蛋白TAP tag-PRAK的表达及细胞内定位情况。结果:重组真核表达载体构建正确,该重组质粒能在HEK293细胞中稳定表达,表达产物TAP tag-PRAK主要分布在核内。结论:成功构建pNTAP-PRAK真核表达载体并建立了其稳定表达的HEK293细胞系,TAP标签未对PRAK定位产生明显影响。AIM： To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification （ TAP）-tagged PRAK. METHODS： Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK,then DH5α E. coli was transformed with the recombinant plasmid. After identified by PCR,digestion with restriction endonuclease and sequencing,the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS： All the results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION： The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didn＇t influence the localization of exogenous PRAK.

关 键 词：p38调节活化蛋白激酶 克隆 分子 基因表达 

分 类 号：Q78[生物学—分子生物学]