IFN-λ真核表达载体的建立及其表达产物的生物学功能评估

摘要

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.%目的：构建 IFN-λ真核表达体系，检测 IFN-λ基因在真核细胞中的表达，评估真核细胞表达 IFN-λ的抗增殖和抗病毒生物学作用。方法从 poly I∶C 刺激的 HuH-7细胞 mRNA 中克隆 IFN-λ1／2基因全长，并进行 DNA 基因测序；构建 pcDNA3-IFN-λ1／2质粒，酶切鉴定，并将其转染 COS-7细胞，应用 Western 印迹检测 IFN-λ1／2在 COS-7细胞中的表达。COS-7细胞表达的 IFN-λ1／2作用于人食管癌 YES5和 T．Tn 细胞株，CCK-8法检测癌细胞增殖， Western 印迹检测凋亡蛋白 caspase-3的活化，实时荧光定量 PCR 检测 ISG15及 MxA 抗病毒基因表达。结果经PCR、DNA 测序和酶切鉴定，克隆的 IFN-λ1／2基因与 GenBank 公布的序列一致，IFN-λ1／2基因序列正确构建入pcDNA3载体中；在 pcDNA3-IFN-λ1／2转染的 COS-7细胞中检测到 IFN-λ1／2蛋白表达；其表达产物可诱导食管癌细胞增殖抑制、活化凋亡蛋白 caspase-3，并且上调抗病毒 ISG15及 MxA 基因。结论建立了 IFN-λ真核表达体系，即通过 pcDNA3-IFN-λ1／2转染 COS-7细胞实现了 IFN-λ表达；经此体系表达的 IFN-λ具有抗增殖、诱导凋亡及上调抗病毒基因表达的生物学作用；此体系的建立为 IFN-λ的生物学功能研究和临床应用奠定了基础。