Construction of pGL3-SM22-SCAP (D443N) eukaryotic expression vector and its expression in CHO cells / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 114-120, 2010.
Article
in Chinese
| WPRIM
| ID: wpr-336253
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Physiology
/
Recombinant Proteins
/
RNA, Messenger
/
Mice, Transgenic
/
Transfection
/
Cricetulus
/
Promoter Regions, Genetic
/
CHO Cells
/
Intracellular Signaling Peptides and Proteins
/
Mutant Proteins
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2010
Type:
Article
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