Cloning Of Swine Gene Ccl17and Its Expression In Prokaryotic And Eukaryotic Cells

Chemokines are generated by a wide variety of cells and function as activators and chemoattractants for leukocytes while the organism is defending and eliminating invading pathogen as well as other non-self molecules. Chemokines also act as a chemoattractant to guide the migration of cells. Cells that are attracted by chemokines follow a signal of increasing chemokine concentration towards the source of the chemokine, thereby immune cells are attracted to the site of immune response and participate in immunomodulation and immunopathological reactions. Finally, chemokine superfamily play significant roles in both the physiological and pathological modulation especially in the autoimmune and inflammatory diseases.Chemokine ligand 17, aka( thymus and activation-regulated chemokine ,TARC) is the first recognized member in CC subfamiliy with super selective chemotactic capability to recruit Th2 cells from peripheral blood to the site of inflammation. Current perspectives focused on the role of CCL17 and its receptor in autoimmune diseases. However, the pathogenesis and immune mechanism of CC17 remains to be explained thoroughly. This study has cloned the porcine CCL17 gene and performed both prokaryotic and eukaryotic expression. The results are as follows.(1)We obtained an EST sequence with accession number of DB794536 from porcine EST database in NCBI. Using blast tools and ORF Finder on NCBI, an ORF of this sequence containing the intact CCL17 gene was found. Designing primers based on this sequence, we cloned porcine CCL17 gene using RT-PCR method and validated the existence of this gene. Results suggested that the ORF contained 309 nucleotides, coding 102 amino acids.(2)CCL17 gene was amplified and cloned to its prokaryotic vector pET-32a(+), and then the recombinant plasmids were transformed to E.Coli BL21 (DE3). By the challenge of variant concentrations of IPTG and other conditions, SDS-PAGE analysis showed that the target protein was overexpressed in E. coli BL21 (DE3) with the optimal condition of 28℃, 0.5mol/L IPTG and 4-6 induction hours(3)CCL17 fragment was ligated into eukaryotic expression vector pEGFP-C1 which contained an enhanced green fluorescence reporter gene, thus recombinant plasmid pEGFP- CCL17 was constructed. The recombinant plasmid and the control plasmid pEGFP-C1 were respectively transfected to SUVEC cells by Lipofectamine 2000 Regent. Positively transfected clones were observed with inversion fluorescence microscope, and CCL17 gene expression were further verified by western-blot. From the results above, conditions and profiles of CCL17 gene expression in vitro were acquired preliminarily. And the findings may offer some references to the further revealing of its immunological properties, detection kit preparation and functional exploration.