CCL21-CD40L Fusion Gene Augment Antitumor Activity

Objective:Dendritic cell is the most functional antigen presenting cells in vivo,it can capture,processing,presenting tumor antigen to T cells,initiating immune response. However,DC vaccine is hard to develop because of its long processing,short survival time and difficulty in mimicing its natural maturation.We construct eukaryotic expression vector carried CCL21-linker-exCD40L fusion gene,test its chemotactic function,and investigate its anti-tumor activity.Methods:Search the gene coding sequence of mouse CCL21 and CD40L in GeneBank, and determine the amplified region.The total mRNA was extracted from mouse spleen using trizol,and the aimed segment were amplified from total mRNA by the method of reverse transcription polymerase chain reaction.CCL21-linker-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L,then it were inserted into pMD18-T for seqencing.Eukaryotic expression plasmid pcDNA3.1 was used to carried the CCL21-linker-exCD40L fusion gene after confirmation of sequencing.We also cloned CCL21 and CCL21sig-CD40L into pcDNA3.1 as comparision.The recombinant plasmid were respectively transfected into Chinese Hamster Ovary(CHO) cells and wsestern blot was used to identify the fusion protein.The chemotatic function of the expressed product was detected by chemotaxis assay and the anti-tumor activity of it was tested with vivo transfection.Result:Gene sequencing,gel electrophoresis of PCR product and restrictive digestion proved the successful construction of pcDNA3.1/CCL21,pcDNA3.1/CCL21sig-exCD40L and pcDNA3.1/CCL21-linker-exCD40L,and its expression was conformed by western blot, which is 14KD,30KD,43KD respectively.We test that the fusion protein had a significant chmotactic function to DCs using Millicell-Cabinet Method,which the cell numbers passing through the film is 14.95 times of blank control every high power microscope visual field. We construct colon cancer model in Balb/c mouse with C26 cell line,and after tumor orthotoic injection of plasmid carrying fusion gene,the expressed product in vivo act anti-tumor function.Conclusion:The recombinant murine CCL21-linker-exCD40L,CCL21 and CCL21sig-exCD40L eukaryotic expression plasmid was successfully constructed,and they all can be expressed by eukaryotic cell after transfection.The expression product of CCL21-1inker-exCD40L fusion gene in tumor cells had a remarkable function to DCs.After tumor orthotopic injection,it can inhibit tumor growth and prolong the mouse survival time, which is more effective than all the control.