Construction and identification of recombinant adenovirus containing heat shock protein70 gene driven by carcinoembryonic antigen promoter
10.3760/cma.j.issn.1674-1935.2011.04.007
- VernacularTitle:CEA启动子驱动下表达Hsp70基因的重组腺病毒的构建及鉴定
- Author:
Yunliang SUN
;
Can XU
;
Changqing SU
;
Jun GAO
;
Jing JIN
;
Hongyu WU
;
Zhaoshen LI
- Publication Type:Journal Article
- Keywords:
Recombination,genetic;
Heat shock protein 70;
CEA promoter;
Adenoviruses
- From:
Chinese Journal of Pancreatology
2011;11(4):251-254
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant adenoviros containing heat shock protein70 (Hsp70) gene driven by carcinoembryonic antigen (CEA) promoter. Methods Hsp70 gene and CEA promoter were amplified by RT-PCR and PCR, and then subcloned into the shuttle vector pDC316 to construct the recombinant vector PDC316-pCEA-Hsp70. The recombinant vector was co-transfected with adenoviral backbone plasmid into HEK293 cells to generate the recombinant adenovirus Ad5-pCEA-Hsp70. The recombinant adenovirus was purified by CsCl banding and titrated by 50％ tissue culture infective dose (TCID50) assay. After transfection of the recombinant adenovirus into human pancreatic cell lines SW1990 and BxPC3, the expression of mRNA and protein level of Hsp70 were determined by RT-PCR and ELISA,respectively. Results Digestion and DNA sequencing certified that the Hsp70 gene and CEA promoter was successfully inserted into pDC316 plasmid. Virus acquired through co-transfection with backbone plasmid was confirmed to be constructed successfully by PCR amplification. The particles finally expressed was 2.2 ×1011vp/ml, and the titer was 1.5 x 1010 PFU/ml. BxPC3 cancer cells with positive CEA expression showed increased expression of Hsp70 mRNA and protein after infected by recombinant adenovirus; while SW1990 cancer cells with negative CEA expression showed no change of expression of Hsp70 mRNA and protein after infected by recombinant adenovirus. Conclusions The recombinant adenovirus Ad5-pCEA-Hsp70 which can express Hsp70 gene in CEA positive cancer cells is constructed successfully.
