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Published on: January 20, 2011
[Construction of recombinant plasmid pIRES2-EGFP/CCK and its expression in vivo and in vitro]
Ji-Gang Bai1, Yia Lü, Hao-Hua Wang
1Department of General Surgery, First Hospital of Xi'an Jiaotong University, Xi'an 710061, China.
Objective:
To construct eukaryotic expression plasmid of porcine CCK gene pIRES2-EGFP/ CCK and express it in COS-7 cells and hamsters.
Methods:
The aimed segments were obtained from intermediate vector pMD18-T/CCK by the method of restricted enzymatic resection and were inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP/CCK. The recombinant expression plasmid was transfected into COS-7 cells by liposome-mediated gene transfer method and observed through Fluorescence microscopy. The plasmid was injected into the skeletal muscle of hamsters directly to detect the expression of the recombinant plasmid in vivo.
Results:
A recombinant eukaryotic expression plasmid pIRES2-EGFP/CCK was successfully constructed. Green fluorescent protein could be detected in the transfected COS-7 cells 24, 48, and 72 hours post transfection and the expression of green fluorescent protein reached its peak 72 h post transfection. The green fluorescent protein could be detected at the injection site on the 4th day post injection and the fluorescence intensity became stronger on the 14th day. The level of fluorescence became ever stronger on the 42nd day. No expression of green fluorescence was detected in the control group.
Conclusion:
Porcine CCK cDNA eukaryotic expression plasmid pIRES2-EGFP/CCK has been successfully constructed and expressed in mammal cells COS-7 and hamster in vivo. The research paved the way for cross immunity therapy of hamster pancreatic carcinoma.
