Construction of recombinant adenovirus vectors carrying proapoptotic gene bimS.
- Author:
Yaning ZHAO
1
;
Qiang LI
;
Haiyan WEI
;
Kun YANG
;
Suju HAO
Author Information
1. Department of Hematology, The Second West China Hospital, Sichuan University, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Adenoviridae;
genetics;
metabolism;
Apoptosis Regulatory Proteins;
biosynthesis;
genetics;
Genetic Therapy;
Genetic Vectors;
genetics;
metabolism;
HEK293 Cells;
HL-60 Cells;
Humans;
Neoplasms;
therapy;
Recombinant Proteins;
biosynthesis;
genetics
- From:
Journal of Biomedical Engineering
2007;24(6):1367-1372
- CountryChina
- Language:Chinese
-
Abstract:
In order to further study the anti-tumor activity of bimS and the feasibility of using adenovirus vector for gene therapy,we constructed a recombinant adenovirus vector of pro-apoptotic factor bimS(pAdEasy-CMV-bimS). Human bimS gene was amplified from HL-60 leukemic cell by RT-PCR and it was identified by sequencing. Then bimS was cloned into the shuttle vector pAdTrack-CMV that carried a green fluorescence protein (GFP) gene to generate a recombinant plasmid pAdTrack-CMV-bimS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E. coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restriction endonuclease digestion. The recombinated adenovirus pAdEasy-CMV-bimS was transferred into HEK293 cell for packaging and amplification. The virus titre was determined and the insert of bimS gene was verified by PCR method. Finally, HEK293 cell was infected by recombinated adenovirus pAdEasy-CMV-bimS (MOI=100) and bimS protein was detected by western blot. GFP expression in transfected HEK293 cells could be observed at 48-72 hours. bimS gene was detected in cultural supernatant of infected HEK293 cell by PCR and there was typical cytopathic effect(CPE) in HEK293 cell 7 days after infection. Western blot showed bimS protein expression in infected HEK293 cells was markedly higher than that in uninfected HEK293 cells. The result indicated that human bimS recombinant adenovirus was constructed successfully, which could therefore provide a sound base for anti-tumor gene therapy with bimS in vivo and in vitro.
