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Journal of Chinese Integrative Medicine ›› 2004, Vol. 2 ›› Issue (1): 42-45.doi: 10.3736/jcim20040116

• Original Experimental Research • Previous Articles     Next Articles

Effect of recombinant adenoviruses with CD/TK fusion suicide gene on human hepatocellular carcinoma cells

Chang-jun Wang1, Jian-jun Li2, Xiao-min Wen3, Li Deng1   

  1. 1. Department of Oncology, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong Province 510130, China
    2. Department of Integrated Traditional Chinese and Western Medicine, Second Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province 510120, China
    3. Department of Traditional Chinese Medicine, First Military Medical University, Guangzhou, Guangdong Province 510515, China
  • Online:2004-01-20 Published:2018-10-25

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Objective: To investigate gene-therapy for human hepatocellular carcinoma with adenovirus vectors by double suicide gene CD/TK. Methods: Double suicide gene CD/TK was liberated from eukaryotic vectors pCEA-CD/TK and subcloned into shuttle vectors, and the transfer plasmid pAdtrack-CMV-CD/TK was formed after linearizing with Pac 1. It was recombinated with pAdeasy-1 in bacteria BJ5183. The identified adenovirus plasmid was digested by Pac1 and was transfected into 293 cells to pack the adenoviruses. After PCR determination, its titre was measured, and the infection rate and efficacy were tested in human hepatocellular carcinoma cells. Results: pAdtrack-CMV-CD/TK and pAd-CD/TK were tested by endonuclease digestion. Ad-CD/TK was produced in 293 cells, and the human hepatocellular carcinoma cells (SMMC7721) infected by Ad-CD/TK were killed after 5-FC was used, and bystander effects were observed.Conclusion: Recombinant adenoviruses with CD/TK fusion suicide gene have a high infection rate and efficacy for human hepatocellular carcinoma cells.

Key words: adenovirus vector, gene therapy, double suicide gene, carcinoma, hepatocellular

CLC Number: 

  • R735.7

Fig.1

Electrophoresis identification of shuttleplasmid pAdtrack-CMV-CD/TK1:pCEA-CD/TK;2:pCEA-CD/TK HindⅢ/EcoRⅠdouble digestion （including 2.5 kb fragment of aimed genes and 5.4 kb fragment of carrier）;3:pCEA-CD/TK HindⅢ/BamHⅠdouble digestion（including about 1.3 kb fragment of CD and 6.6 kb fragment）;4:pAdtrack-CMV-CD/TK BamHⅠdigestion（12 kb）;5:pAdtrack-CMV-CD/TK HindⅢ/BamHⅠdouble digestion（6 kb,5 kb,1.3 kb）;M:DNA marker"

Fig.2

Electrophoresis identification of rAdplasmid pAd-CD/TK1:pAdtrack-CMV-CD/TK HindⅢ/BamHⅠdouble digestion（6 kb,5 kb,1.3 kb）;2:pAd-CD/TK PacⅠdigestion （30 kb and 4.5 kb）;3:pAdeasy-1 PacⅠdigestion（33 kb）;4:pAd-CD/TK HindⅢ/BamHⅠdouble digestion（including 1.3 kb fragment of CD）;M:DNA marker"

Fig.3

PCR identification of rAd plasmid Ad-CD/TK1:negative control;2:rAd （1.3 kb）;M:DNA marker"

Fig.4

Fluorescence photo of 293 cells afterAd-CD/TK infection"

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