柯萨奇-腺病毒受体不同胞内功能域缺失突变载体的构建与表达  

Construction and expression of different intracellular functional-domain deleted recombinant vector of coxsackie-adenovirus receptor

作　　者：范良生[1] 王鸿雁[1] 王蓓蓓[1] 陈刚[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肿瘤生物医学中心,武汉430030

出　　处：《广东医学》2011年第15期1945-1949,共5页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:30700895;30672227;30770913);科技部"973"计划资助项目(编号:2009CB521808)

摘　　要：目的以柯萨奇-腺病毒受体(Coxsackie and adenovirus receptor,CAR)蛋白结构的功能解析作为切入点,构建不同CAR胞内域缺失突变的表达载体。方法利用定点缺失突变策略构建CAR胞内域PKC、CK2、TYR磷酸化位点的缺失突变体及胞内域完全缺失的突变体。利用三步PCR法扩增获得相关的基因片段后,将其克隆入真核表达载体pcDNA3.1/V5/His,通过双酶切及测序进行鉴定后,分别转染至低表达CAR的人卵巢癌细胞系SKOV3细胞中,并用Werstern blot检测细胞转染后CAR的表达水平。结果所有突变体经过测序分析均证实序列与设想的突变序列相符,各突变体在SKOV3细胞中获得了满意的表达。结论成功构建不同CAR胞内功能域缺失突变表达载体,为后续研究CAR及其胞内不同功能域在卵巢癌中的作用机制提供了研究基础。Objective To construct different intracellular functional - domain deleted recombinants of Coxsackie - adenovirus receptor （CAR） for functional analysis. Methods Recombinant pcDNA3. 1/VS/His - ACK2, pcDNA3. 1/ VS/His - APKC, peDNA3, 1/VS/His - ATYR and pTOPO - tailless were constructed amplified on the basis of three - step deletion mutation PCR. The recombinants were transfected into ovarian cancer cell line SKOV3, in which low CAR was expressed, by Lipofectamine 2000. Their protein expression levels were detected by western blot. Results All the mutated sequences were consistent with our envisagement confirmed by DNA sequencing and restriction analysis. Further- more, CAR expression was up -regulated in SKOV3 transfected with recombinants. Conclusion Recombinants provide important groundwork in the study of molecular mechanisms of different functional domains of CAR in ovarian cancers.

关 键 词：柯萨奇-腺病毒受体 功能域缺失突变重组体 卵巢癌 

分 类 号：R737.11[医药卫生—肿瘤]