Construction and expression of recombinant adenovirus vector Ad5-CCL20 / 中国现代普通外科进展

ABSTRACT

Objective:To construct recombinant adenovirus vector Ad5-CCL20,and detect the expression of CCL20 after Ad5-CCL20 transfected colon cancer cells CT-26.Methods:Genes encoding CCL20 was obtained from original plasmid double-digested with EcoR I/Sal I enzymes.The CCL20 DNA segments were linked into pDC316 to recombine shuttle plasmid pDC316-CCL20.After genome sequencing,we take shuttle plasmid pDC316-CCL20 and plasmid backbone pBHGIox_E1,3Cre co-transfecting 293T cells in mediation of liposome.The constructed recombinant adenovirus vector was named Ad5-CCL20.Lastly,after Ad5-CCL20 transfected CT-26 cells in vitro,the expression of CCL20 at different time points (12h,24h,36h and48h)was detected by Western blot and Elisa.Then,Culture supernatant was added into iDC and mDC to evaluate the chemotactic activity of CCL20.Results:The recombinant adenovirus Ad5-CCL20 were successfully constructed.The expression of CCL20 was detected by Western blot and Elisa.The level of CCL20 expression was increased with prolonged incubation of the infected CT-26 cells.Chemotaxis experiments show that the chemokine CCL20 had chemotactic activity to the iDC and mDC,but more obviouly for iDC (P＜0.05).Conclusion:The construction and obtain of recombinant adenovirus vector Ad5-CCL20 provide a new method for developing tumor immunotherapy.