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

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SCAP 443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-time PCR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。

Abstract:

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.