Construction of recombinant adenoviruses containing cytosine deaminase gene driven by the vascular endothelial growth factor promoter using an AdEasier-1 system

Abstract

Abstract: Objective To efficiently construct a recombinant adenovirus containing cytosine deaminase (CD) gene driven by vascular endothelial growth factor (VEGF) promoter using an AdEasier-1 system and observe its killing effect on LoVo cells in vitro. Methods The CD gene was amplified by PCR, and amplicons were cloned in JM109 bacteria, and then recombined into pREP8 to obtain pREP8-CD, which was then digested by HindⅢand XbaⅠfor the CD fragment with polyadenylation site (CD-pA) subcloned into the VEGFP -containing shuttle plasmid pAdtrack-VEGFP to generate pAdtrack-VEGFP-CD-pA. After linearization with PmeⅠ, pAdtrack-VEGFP-CD-pA was transformed into AdEasier-1 cells, and the transformants were selected on LB agar plates containing 25 μg/ml kanamycin followed by identification of the positive pAdEasy-VEGFP-CD with electrophoretic analysis and enzymatic digestion. pAdEasy-VEGFP-CD was then digested with PacⅠand transfected into 293 cells to produce the recombinant adenovirus Ad-VEGFP-CD, which was finally confirmed by PCR. The positive recombinant adenoviruses were transfected into LoVo cells to observe their in vitro anti-tumor effect. Results pAdEasy-VEGFP-CD was constructed with a success rate of 70%. After being packaged in 293 cells and purified by CsCl banding, the titer of the recombinant adenovirus Ad-VEGFP-CD reached as high as 4.8×10¹² CFU particle/ml, and the adenovirus was further confirmed by PCR analysis. In the presence of the prodrug 5-FC, the recombinant adenoviruses remarkably inhibited the growth of LoVo cells. Conclusion The recombinant adenoviruses containing CD gene under the control of VEGF promoter can be efficiently generated using the AdEasier-1 system, and exhibit potent anti-tumor effect in vitro.

CLC Number:

CHEN Jian-fa¹, HUNAG Zong-hai¹, YU Jin-long¹, LI Zhou¹, CHE Xiao-yan². Construction of recombinant adenoviruses containing cytosine deaminase gene driven by the vascular endothelial growth factor promoter using an AdEasier-1 system[J]. Journal of Southern Medical University, 2004, 24(06): 623-627.

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