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

Construction of a eukaryotic expression system of IFN-λ and evaluation of bio-functions mediated by the system product

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Hu H-7细胞mRNA中克隆IFN-λ1/2基因全长,并进行DNA基因测序;构建pc DNA3-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Mx A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Gen Bank公布的序列一致,IFN-λ1/2基因序列正确构建入pc DNA3载体中;在pc DNA3-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Mx A基因。结论建立了IFN-λ真核表达体系,即通过pc DNA3-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。

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＆amp;nbsp;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.