Construction of a eukaryotic expression system of IFN-λ and evaluation of bio-functions mediated by the system product
10.7644/j.issn.1674-9960.2015.11.002
- VernacularTitle: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
- Author:
Jing LI
;
Yixin QI
;
Guiying FANG
;
Yaqi LI
;
Huimin ZHOU
;
Wenxuan LIU
;
Jie HU
;
Quanhai LI
- Publication Type:Journal Article
- Keywords:
IFN-λ;
eukaryotic expression;
anti-proliferation;
apoptosis;
anti-virus
- From:
Military Medical Sciences
2015;(11):816-820
- CountryChina
- Language:Chinese
-
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.
