慢病毒介导的稳定表达鸭干扰素-γ细胞株的构建  

Construction of lentivirus-mediated stable expression ofduck interferon-γ cell line

作　　者：陈柳[1] 倪征[1] 刘可姝 叶伟成[1] 华炯钢[1] 云涛[1] 朱寅初 张存[1] CHEN Liu;NI Zheng;LIU Keshu;YE Weicheng;HUA Jionggang;YUN Tao;ZHU Yinchu;ZHANG Cun(Institute of Animal Husbandry and Veterinary,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,Zhejiang)

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021

出　　处：《浙江农业科学》2024年第1期208-212,共5页Journal of Zhejiang Agricultural Sciences

基　　金：国家重点研发计划(2016YFD0500107-3)。

摘　　要：干扰素-γ是一种具有抗病毒活性和免疫调节能力的细胞因子之一。为了建立稳定表达鸭干扰素-γ(duIFN-γ)的细胞系,本研究优化并合成了duIFN-γ基因,插入到慢病毒表达载体plenti-GIII-CMV-GFP-2A-Puro,获得重组质粒plenti-IFN,将plenti-IFN与辅助质粒共转染包装细胞293T,筛选出了携带duIFN-γ基因的重组慢病毒rlenti-IFN,将该病毒感染中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞,利用有限稀释法和抗性加压筛选法,筛选出了30个具有嘌呤霉素抗性基因的单克隆细胞系。利用RT-qPCR荧光定量法对这些单克隆细胞的duIFN-γ mRNA水平进行检测,筛选出了duIFN-γ mRNA水平最高的1个单克隆细胞,对其进行扩大培养,获得了1株稳定细胞系。Western blot检测结果表明,duIFN-γ稳定表达于该细胞系中。本研究获得了1株稳定表达duIFN-γ蛋白的细胞系,该研究为开展duIFN-γ生物学功能研究、建立duIFN-γ检测方法及生产廉价鸭(禽)用干扰素奠定了基础。Interferon-γis one of the cytokines with antiviral activity and immunomodulatory ability.To construct a cell line stably expressing duck interferon-γ,the gene of duck interferon gamma(dIFN-γ)was optimized referring to mouse codon and synthesized.Synthesed dIFN-γwas cloned into lentiviral expressing vector plenti-GIII-CMV-GFP-2A-Puro to construct recombinant plasmid plenti-IFN.The recombinant lentiviral rlenti-IFN was rescued by co-transfected plenti-IFN and helper plasmid into packaging cell 293T.After infection Chinese hamster ovary(CHO)cells with recombiant rlenti-IFN,30 monoclonal cell lines with purinomycin resistance were selected by limited dilution and pressure screening.At last,one monoclonal cell line with the highest duIFN-γmRNA level was selected by RT-qPCR method.One stable cell line was finally obtained by expanding culture.Western blot analysis indicated that duIFN-γwas stably expressed in this cell line.This study lays a foundation for studying duIFN-γbiological function,establishing duIFN-γdetection method and producing cheap interferon for duck or poultry.

关 键 词：鸭干扰素-γ 慢病毒 CHO细胞 稳定表达 

分 类 号：S858.32[农业科学—临床兽医学]