Construction of pGL3-SM22-SCAP (D443N) eukaryotic expression vector and its expression in CHO cells.
- Author:
Yuanyuan WANG
1
;
Jieli HU
;
Jing CUI
;
Ailong HUANG
;
Xiongzhong RUAN
;
Yaxi CHEN
Author Information
1. Center for Lipid Research, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
- Publication Type:Journal Article
- MeSH:
Animals;
CHO Cells;
Cricetinae;
Cricetulus;
Genetic Vectors;
genetics;
Humans;
Intracellular Signaling Peptides and Proteins;
genetics;
physiology;
Membrane Proteins;
biosynthesis;
genetics;
physiology;
Mice;
Mice, Transgenic;
Microfilament Proteins;
genetics;
Muscle Proteins;
genetics;
Mutant Proteins;
biosynthesis;
genetics;
Promoter Regions, Genetic;
genetics;
RNA, Messenger;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
Transfection
- From:
Chinese Journal of Biotechnology
2010;26(1):114-120
- CountryChina
- Language:Chinese
-
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.
