Construction of eukaryotic expression vector for human CCL21 gene and characterization of its chemotactic function

The eukaryotic expression vector carrying the full length of the human CCL21 gene was constructed in this study and characterization of its chemotactic function was performed in vitro. By using Xho I and BamH I, the human CCL21pSK(hCCL21pSK) was digested, by which a fragment of 540 bp was recovered by agarose electrophoresis and subcloned to the responding site of the eukaryotic expression vector pVAX1. After confirmation by colony PCR and double enzyme digestion analysis, positive recombinant plasmid was transfected into COS-7 cells in vitro by electroporation. Cultural supernatant was collected 72 hours after transfection and the chemotactic function of the expressed products to lymphocytes were detected by chemotaxis assay. Special proteins of CCL21 in cultural supernatants and lysates of transfected cells were detected by Western blotting analysis. By means of colony PCR and restriction enzyme digestion analysis, it was proved that the recombinant expression vector pVAX1-hCCL21 had been successfully constructed in the present study, and the transfected cells expressing the CCL21 protein and the cultural supernatants of these cells showed a potent chemotactic function to lymphocytes. It is concluded that the recombinant eukaryotic expression plasmid pVAX1-hCCL21 has been constructed successfully and to be transiently expressed in COS-7 cells, thus providing a useful murine tumor model for the study of the CCL21 gene therapy.