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.
Abstract
Interferon-γ is 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-γ was 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-γ mRNA level was selected by RT-qPCR method.One stable cell line was finally obtained by expanding culture.Western blot analysis indicated that duIFN-γ was stably expressed in this cell line.This study lays a foundation for studying duIFN-γ biological function,establishing duIFN-γ detection method and producing cheap interferon for duck or poultry.
基金项目
国家重点研发计划(2016YFD0500107-3)
NETL
NSTL
维普
万方数据