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Published on: May 6, 2015
[Construction of a recombinant adenovirus Ad5F35-SD-EGFP and its effect on K562 cell proliferation]
1Department of Clinical Laboratory, First Affiliated Hospital of Chongqing Medical University, Chongqing Medical University, Chongqing 400016, China. sj_677@163.com
Objective:
To construct a recombinant adenovirus vector for SH2-DED fusion gene and assess its inhibitory effect on the proliferation of K562 cells.
Methods:
SH2-DED fusion gene and its mutant SH2mt-DED were amplified by splicing PCR and cloned into pAdTrack-CMV plasmid separately to construct the shuttle plasmids pAdT-SD-EGFP and pAdT-SmD-EGFP, respectively. After Pme I digestion, the shuttle plasmids were transformed into ultra-competent pAd5F35-BJ5183 cells to generate defective adenovirus vectors pAd5F35-SD-EGFP and pAd5F35- SmD-EGFP by homologous recombination. The vectors, linearized by Pac I digestion, were further transfected into AD293 cells for packaging and amplified by infecting AD293 cells repeatedly. K562 cells were then infected by the recombinant adenoviruses and the expression of SD was detected by Western blotting. MTT assay and flow cytometry were used to investigate the effect of Ad5F35-SD-EGFP and Ad5F35-SmD-EGFP on the proliferation of K562 cells.
Results:
The recombinant adenovirus vectors pAd5F35-SD-EGFP and pAd5F35-SmD-EGFP were constructed correctly, with a titer reaching 1.5×10(12) pfu/ml after amplification. Western blotting demonstrated that the target proteins were effectively expressed in transfected K562 cells. MTT assay and flow cytometry showed that transfection with pAd5F35-SD-EGFP resulted in growth inhibition rate of 55.21% in K562 cells, significantly higher than the inhibition rate of 17.95% following transfection with pAd5F35- SmD-EGFP and 7.33% following PBS treatment (P<0.05).
Conclusion:
The recombinant adenovirus vector Ad5F35-SD-EGFP we constructed can significantly inhibit the proliferation of K562 cells in vitro.
