Construction of rat chemokine CCL19 lentivirual vector and expression in vitro

Objective To construct CCL19 expression lentivirus and establish in vitro model of CCL19 expression.Methods The CCL19 gene cDNA was amplified from subclone vector,double cut and linked to construct lentiviral vector.The third generation envelope system was used to produce lentiviral particles.Titers of virus were determined by quantitative polymerase chain reaction (PCR) and protein expression of CCL19 in IEC6 cells was detected by using Western blotting.Rat dendritic cells were isolated and Transwell was used in lentivirus infected IEC6 cells to evaluate chemotaxis.Results PCR and sequencing confirmed that 326 bp cDNA of CCL19 was linked into vector and the rat chemokine CCL19 lentivirual vector was successfully constructed.Titer of lentivirus was determined at 2 × 109 TU/ml via quantitative PCR.Fluorescent observation showed that 80％ expression rate was obtained in IEC6 infection in vitro.Results also demonstrated that Western blotting confirmed CCL19 protein expression in infected rat intestinal epithelial cells.Traswell presented notable increase of dendritic cells net immigration by 4 fold when CCL19 was overexpressed in IEC6 cells.Conclusion CCL19 expression lentivirus was successfully constructed and CCL19 protein over-expression was detected.The chemotaxis of dendritic cells was stimulated by CCL19 overexpression in IEC6 cells. Key words: Dendritic cells; Chemotaxis; CCL19