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《Acta Universitatis Medicinalis Anhui》 2006-01
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Construction and verification of a novel CRAd driven by hTERT promoter

Ni Fang~1, Lu Zhuozhuang~2, Qu Chengkui~1, et al ( ~1Dept of Pathophysiology, Anhui Medical University, Hefei 230032; ~2Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850)  
Objective To construct a novel CRAd vectors driven by hTERT promoter and to establish the foundation for cancer gene therapy. Methods The hTERT promoter as E1A and E1Bp gene were amplified by PCR;the GFP gene and GM-CSF were also amplified by PCR, and the products were subcloned into Teasy plasmid in certain order to generate pSh-GFP-SV55K plasmid. A recombinant adenoviral genomic DNA was obtained by homologous recombinant in E.coli BJ5183 cells pretransformed with the pAdeasy-1 plasmid (BJ5183-AD-1 cells); the recombinant adenovirus Ad-GFP-SV55K was gained after transfecting 293 cell line with the genomic DNA using LepofectAMINE method; PCR was used to identify the recombinant Ad, and the titer of virus was determined by plaque assay in the 293 cells. Results The structure of the adenovirus vectors carrying GM-CSF gene and GFP gene was identified by restriction enzyme analysis. The titer of the virus was 7×10~ 10 pfu/ml. Conclusion A novel recombinant adenovirus vector driven by hTERT promoter is made successfully, and the newly constructed adenovirus is useful for cancer gene therapy.
【Fund】: 国家973课题(编号:2004CB518801);; 国家自然科学基金资助(编号:30470735);; 安徽省人才开发基金资助(编号:2002Z035)
【CateGory Index】: R346
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