Construction Of Human KCNJ15 Gene Eukaryotic Expression Vector And In Vitro Investigation Of Its Function

Background and objectiveSevere hepatitis has been charaterised by rapid deteriation of liver function with a high mortality rate and the underlined mechanisms are not fully understood. Our previous work has illustrated that KCNJ15, an inwardly rectifying potassium channel gene, evidently highly expressed in peripheral blood monocytes (PBMCs) from patients with severe chronic hepatitis B(SHB) when compared with mild chronic hepatitis B (CHB) by microarray analysis. The highly expressed cell types were identified to be CD4+ T cells and CD8+ T cells, especially CD4+ T cells. Thus in this current study we constructed human KCNJ15 gene eukaryotic expression vector and then tansfected it into Jurkat T cell line (human T Lymphocyte from acute T cell leukemia) by electroporation to further study its role in activation of T cells.MethodsHeparinized human peripheral venous blood was obtained from consent informed patients with severe chronic hepatitis B (SHB). After isolation of PBMCs, the mRNA was extracted and then reverse transcripted to cDNA. By specific primers, the open reading frame(ORF) of human KCNJ15 was amplificated and cloned into pcDNA3.1(+) eukaryotic expression vector. The sequence of human KCNJ15 was confirmed by further sequencing. The KCNJ15 expression plasmid was then trasfected into Jurkat T cell by electroporation. The gene and protein expression was measured by Real-time PCR and Western-blot analysis. T cell activation was identified by its surface activation markers CD25 and CD69 as well as cytokines IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, IL-17A, and IL-9 production by Cytometric Bead Array.ResultsHuman KCNJ15 eukaryotic expression vector was successfully constructed and transfected into Jurkat T cell. Though non-significant expression of activation markers CD25 and CD69 was observed, elevated expression of IL-17A and decreased production of IL-9 were evidenced in trasfected T cells, whereas IL-2, IL-4, IL-6, IL-10, TNF, IFN-γproduction remained unchanged.ConclusionThe preliminary data presented here shown that human inwardly rectifying potassium channel gene KCNJ15 contributes to T cell activation by increased IL-17A secretion and decreased IL-9 production in vitro. These results provided a basis for further investigation of KCNJ15's role in T cell activation in vivo and eventually contribution to immune mediated hepatocyte injury in patients with SHB.