SM22启动SCAP真核表达质粒的构建及其在CHO细胞中的表达  

Construction of pGL3-SM22-SCAP (D443N) eukaryotic expression vector and its expression in CHO cells

作　　者：王媛媛[1] 胡接力[1] 崔静[1] 黄爱龙[1] 阮雄中[1,2] 陈压西[1] 

机构地区：[1]重庆医科大学附属第二医院教育部感染性疾病分子生物学重点实验室脂质研究中心,重庆400016 [2]Center for Nephrology,Royal Free and University College Medical School,University College London,Royal Free Campus,London,UK

出　　处：《生物工程学报》2010年第1期114-120,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(Nos.30772295;30871159;30971389;30530360);重庆市自然科学基金(No.2008BA5016)资助~~

摘　　要：为建立平滑肌特异的固醇调节元件结合蛋白(SREBP)的裂解激活蛋白(SCAP)超表达的转基因小鼠,深入探讨SCAP的功能,本实验构建了由平滑肌特异蛋白SM22启动子(pSM22)启动仓鼠SCAP443位点突变体——SCAP(D443N)的真核表达质粒,并在仓鼠卵巢细胞(CHO)验证其表达。利用巢式PCR从小鼠肝脏组织提取的基因组中扩增得到pSM22基因。先将其插入pMD-T载体,构建T-SM22,对pSM22测序后,通过双酶切将pSM22克隆到pGL3-control-Luc中,成为pGL3-SM22-Luc。转染pGL3-SM22-Luc到血管平滑肌(VSMCs)中,通过检测荧光素酶(Luc)值观察pSM22在VSMCs内的启动活性。利用PCR从pTK-HSV-SCAP(D443N)质粒中扩增出SCAP(D443N)后克隆入pGL3-control中,成为pGL3-SCAP。然后再将pSM22克隆入pGL3-SCAP中,成为表达质粒pGL3-SM22-SCAP(D443N)。转染表达质粒到CHO细胞,用real-timePCR和Western blotting验证SCAP(D443N)的表达。结果证实pSM22在体外VSMCs中能启动Luc的表达;表达质粒pGL3-SM22-SCAP(D443N)酶切及测序结果正确;将其转染到CHO细胞后,与转染pGL3-control的对照细胞相比SCAP(D443)mRNA和蛋白表达显著增强。The experiment was designed to investigate the function of SREBP cleavage-activating protein （SCAP） mutant （D443N） by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 （pGL3-SM22-SCAP（D443N））. SM22 promoter （pSM22） was amplified from genome DNA of mice by nested PCR, and then cloned into pMD-T vector. The SM22 promoter fragment released from the vector by Kpn I and Hind III digestion was sub-cloned into pGL3-control-Luc vector, to form pGL3-SM22-Luc. The activity of pSM22 in human vascular smooth muscle cells （VSMCs） was tested using Dual-Luciferase Reporter System. SCAP（D443） mutant amplified from plasmid pTK-HSV-SCAP（D443N） and pSM22 from mice liver were cloned into pGL3-control vector to construct pGL3-SM22-SCAP（D443N） which was transfected into Chinese hamster ovary cells （CHO） to test SCAP（D443） expression by real-time PCR and Western blot. The sequence and construction of pGL3-SM22-SCAP（D443N） were correct. SM22 promoter activity initiated the expression of luciferase in VSMCs and also drove SCAP（D443） expression in transfected CHO cells. The pGL3-SM22-SCAP（D443N） eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.

关 键 词：SM22 启动子 SREBP裂解激活蛋白 质粒构建 基因表达 转染 

分 类 号：Q78[生物学—分子生物学]