[Construction of recombinant plasmid pIRES2-EGFP/CCK and its expression in vivo and in vitro].
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 and were inserted into an 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 was observed through fluorescence microscope. 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 after the transfection. On the 4th day postinjection into the skeletal muscle of hamsters, the protein could be detected at the injection site and the fluorescence intensity became much stronger on the 14th day than that on the 4th day. On the 42nd day the protein level increased. The green fluorescence protein was never expressed in the untransfected cells.


CONCLUSIONS
The porcine CCK gene eukaryotic expression plasmid pIRES2-EGFP/CCK is constructed successfully, and is expressed in mammal COS-7 cells and hamsters in vivo. The research paves the way for the cross immunity therapy of hamster pancreatic carcinoma.
Department of General Surgery, First Hospital of Xi' an Jiaotong University, Xi' an 710061, China.
Yi
Y
Lu
Ji-gang
JG
Bai
Hao-hua
HH
Wang
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D019496:Cancer Vaccines / Q000627:therapeutic use; D002522:Chlorocebus aethiops; D002766:Cholecystokinin / Q000096:biosynthesis* / Q000235:genetics; D006224:Cricetinae; D005057:Eukaryotic Cells / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000628:therapy; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013552:Swine; D014162:Transfection
D019496:Cancer Vaccines; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002766:Cholecystokinin