鸭γ干扰素在体外细胞中抗鸭肠炎病毒作用的研究

Study on effect of duck gamma interferon against duck enteritis virus on cells in vitro

为初步探究鸭γ干扰素（IFNγ）抗病毒作用,本研究从鸭胚成纤维细胞（duck embryo fibroblast,DEF）中扩增出鸭γ干扰素（IFNγ） cDNA。将鸭IFNγ基因分别与真核表达载体PCAGG及原核表达载体p ET-32a连接,获得重组质粒PCAGG-IFNγ及pET-32a-IFNγ。转染PCAGG-IFNγ于DEF细胞后第24小时,接种鸭肠炎病毒（duck enteritis virus,DEV）。同时将pET-32a-IFNγ在大肠杆菌原核表达系统中进行蛋白表达,并用纯化产物处理DEF细胞,处理后第24小时接种DEV,分别采用Western-blot、TCID50测定方法和荧光定量PCR检测重组蛋白IFNγ对DEV复制的影响,并检测细胞中相关干扰素刺激因子（interferonstimulated genes,ISGs）基因包括抗黏液病毒基因（Myxovirus resistance,Mx）、泛素样2′-5′寡聚腺苷酸合成酶基因（2′-5′oligoadenylatesynthetases-like,OASL）、DEAD框蛋白50基因（DEAD-box protein 50,DDX50）的表达情况。结果发现,无论是真核表达的还是原核表达的重组IFNγ均能抑制DEV的复制,并引起ISGs基因表达的上调。本研究结果为进一步探索鸭IFNγ的抗病毒机制提供了基础数据。

In order to preliminarily explore the antiviral effect of duck IFNγ,duck IFNγ c DNA was amplified from duck embryo fibroblast（DEF）.The duck IFNγ gene was ligated with the eukaryotic expression vector PCAGG and theprokaryotic expression vector p ET-32 a respectively to obtain the recombinantplasmids PCAGG-IFNγ and p ET-32 a-IFNγ.PCAGG-IFNγ was transfected into DEF cells for 24 hours,then duck enteritis virus（DEV） was inoculated.At the same time,p ET-32 a-IFNγ was expressed in Escherichia coli prokaryotic expression system,and DEF cells were treated with the purified producttion,then DEV were inoculated for 24 hours.Western blot,TCID50 and fluorescence quantitative PCR were used to detect the effect of recombinant protein IFNγ on DEV replication,and the expression changes of interferon-stimulated genes（ISGs）such as Myxovirus resistance（Mx）,2＇-5＇ oligoadenylatesynthetases-like（OASL）,and DEAD-box protein 50（DDX50）also were detected.The results showed that both eukaryotic expression and prokaryotic expression of recombinant IFNγ inhibited the replication of DEV and caused the up-regulation of ISGs expression. This study provides basic data for further study on the antiviral mechanism of duck IFNγ.