慢病毒介导的稳定表达鸭干扰素-γ细胞株的构建

doi:10.16178/j.issn.0528-9017.20218494

浙江农业科学 ›› 2024, Vol. 65 ›› Issue (1): 208-212.DOI: 10.16178/j.issn.0528-9017.20218494

慢病毒介导的稳定表达鸭干扰素-γ细胞株的构建

陈柳(), 倪征, 刘可姝, 叶伟成, 华炯钢, 云涛, 朱寅初, 张存()

浙江省农业科学院畜牧兽医研究所,浙江杭州 310021

收稿日期:2023-02-22 出版日期:2024-01-11 发布日期:2024-01-17
通讯作者: 张存(1965—),男,安徽庐江人,研究员,硕士,主要从事水禽病毒研究,E-mail:zhangcun@aliyun.com。
作者简介:陈柳(1980—),女,湖北天门人,副研究员,博士,主要从事动物病毒分子生物学研究,E-mail:haoliuzi@126.com。
基金资助:
国家重点研发计划(2016YFD0500107-3)

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

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

Received:2023-02-22 Online:2024-01-11 Published:2024-01-17

摘要/Abstract

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。

关键词: 鸭干扰素-γ, 慢病毒, CHO细胞, 稳定表达

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.

Key words: duck interferon-γ, lentivirus, CHO cell, stable expression

中图分类号:

S858.32

陈柳, 倪征, 刘可姝, 叶伟成, 华炯钢, 云涛, 朱寅初, 张存. 慢病毒介导的稳定表达鸭干扰素-γ细胞株的构建[J]. 浙江农业科学, 2024, 65(1): 208-212.

CHEN Liu, NI Zheng, LIU Keshu, YE Weicheng, HUA Jionggang, YUN Tao, ZHU Yinchu, ZHANG Cun. Construction of lentivirus-mediated stable expression of duck interferon-γ cell line[J]. Journal of Zhejiang Agricultural Sciences, 2024, 65(1): 208-212.

图/表 3

图1 重组plenti-IFN质粒的EcoR V酶切鉴定

Fig.1 Identification of recombinant plenti-IFN by EcoR V digestion

图2 重组慢病毒rlenti-IFN感染的CHO细胞 a、b—重组病毒rlenti-IFN;c—细胞克隆。

Fig.2 CHO cells infected with rlenti-IFN

图3 Western blot方法检测稳定细胞系CHO-IFN中IFN-γ的表达 1—对照,CHO-lenti-CN细胞;2—CHO-IFN细胞。

Fig.3 Detection of IFN-γ expression in stable cell line CHO-IFN by Western blot

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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 of duck interferon-γ cell line

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