ABSTRACT
Objective To construct a eukaryotic expression system of IFN-λ,examine the expression of IFN-λand evaluate its bio-functions including anti-proliferation and anti-viral activity.Methods The genes of human IFN-λ1 /2 (hIFN-λ1 /2)were cloned from the mRNA of poly I∶C treated HuH-7 cells.The PCR product was examined with DNA sequencing.The genes of IFN-λ1 /2 were sub-cloned into pcDNA3 vector.The correct insertion of the gene IFN-λ1 /2 was identified with enzyme digestion.The constructed pcDNA3-IFN-λ1 /2 plasmids were transfected into COS-7 cells and IFN-λ1 /2 protein was checked in the supernatant and lysis of transfected cells using Western blotting analysis.The human esophageal carcinoma YES5 and T.Tn cells were treated with the IFN-λ1 /2 from the transfected cells and the proliferation of carcinoma cells were measured with CCK-8 kit.In the treated carcinoma cells,the apoptosis and antivirus related molecules such as caspase-3,ISG15 and MxA was analyzed with Western blotting or Quantitative real time PCR.Results The sequence of hIFN-λ1 /2 fragment matched that of the gene bank and the gene of the cytokines was inserted into pcDNA3 vector correctly.With Western blotting analysis,IFN-λ1 /2 protein was detected in the pcDNA3-IFN-λ1 /2 transfected COS-7 cells.The IFN-λ1 /2 from the transfected COS-7 cells inhibited the growth of YES5 and T.Tn cells, activated apoptosis related caspase-3,and up-regulated the anti-virus gene expression of ISG15 and MxA.Conclusion COS-7 cells can express IFN-λ1 /2 after transfection with pcDNA3-IFN-λ1 /2,suggesting that eukaryotic expression system of IFN-λis established.IFN-λ1 /2 from the system can perform bio-functions,such as proliferation inhibition,apoptosis induction and anti-viral gene up-regulation,which indicates that the system can contribute to further investigations of IFN-λbio-activity and its clinical application.
ABSTRACT
Objective To investigate the biological function of IFN-λ in 7 human esophageal carcinoma cells. MethodsThe gene expression of IL-28α, IL-10β and antiviral molecule was examined with PCR. The MHC molecules expression and the profiles of cell cycle were analyzed with flow cytometer. Cell proliferation was evaluated with MTT assay. ResultsAll of esophageal carcinoma cells express the gene of II-28α and IL-10β. IFN-λ induced or augmented the gene expression of antiviral molecules, 2′5′-OAS and MxA. IFN-λ enhanced the MHC class Ⅰ molecule expression. IFN-λ inhibited the growth of esophageal carcinoma cells through the regulation of cell cycle distribution. ConclusionEsophageal carcinoma cells express the IFN-λ receptor complex. IFN-λ has the antiviral, anti-proliferative and immunoregulation activity.