Construction and verification of replication-deficient recombinant adenovirus to silence CTGF gene by AdEasy system
10.3781/j.issn.1000-7431.2010.03.005
- VernacularTitle:沉默结缔组织生长因子基因重组腺病毒的构建及功能验证
- Author:
Rui LIANG
;
Quan KANG
;
Junjie TAN
;
Lihua ZHAO
;
Taoli SUO
;
Yanhui SUN
;
Xianqing JIN
;
Qing LUO
- Publication Type:Journal Article
- Keywords:
Adenoviridae infections;
DNA,recombinant;
RNA interference;
Gene,CTGF;
Cell,4T1
- From:
Tumor
2010;(3):194-198
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To design and construct the replication-deficient recombinant adenovirus Ad-siCTGF which can silence the expression of connective tissue growth factor (CTGF) by RNA interference and verified its function. Methods:A specific sequence, which was verified to be able to silence CTGF gene with high efficiency, was cloned into pSES-HUS vector to produce the shuttle plasmid pSES-siCTGF. The plasmid after Pme Ⅰ linearization was cotransduced with pAdEasy into BJ5183 E.coli strains to construct recombinant vector Ad-siCTGF. After linearization treatment with Pac Ⅰ enzyme digestion Ad-siCTGF was transfected into HEK293 cells via liposome mediation. The recombinant adenovirus was packaged. The titer of the Ad-siCTGF was increased after three times of cross-infection. 4T1 cells were infected with the adenovirus. The silencing efficiency was tested by real-time fluorescence quantitative (RFQ)-PCR and Western blotting.Results:Pac Ⅰ enzyme digestion electrophoresis indentified that recombinant adenovirus was successfully constructed. The titer of the recombinant adenovirus Ad-siCTGF was 2.6×10~(10) pfu/mL after amplification and purification. The CTGF mRNA and protein expression in 4T1 cells were decreased by 36.27% and 31.56%, respectively, compared with the control groups.Conclusion:The recombinant adenovirus which can silence the expression of CTGF was successfully constructed. It laid a good foundation for further investigation of the action mechanism of CTGF in tumor cells.
