荣昌猪γ干扰素重组杆状病毒的构建及其抗病毒活性分析
Construction and Antiviral Activity Analysis of Rongchang Porcine Interferon-γ Recombinant Baculovirus
摘要: 本试验通过扩增、克隆荣昌猪的IFN-γ基因，利用Bac-to-Bac杆状病毒/昆虫细胞表达系统构建含有荣昌猪IFN-γ完整开放阅读框的供体质粒pFastBacTM Dual-IFN-γ，转化至DH10Bac感受态细胞，获得杆状病毒重组穿梭质粒Bacmid-IFN-γ，转染对数生长期的sf9昆虫细胞得到重组杆状病毒rBac-IFN-γ。在有感染重组杆状病毒的猪肾细胞(PK-15)上接种TGEV后观察细胞病变情况并利用PT-PCR技术对rPoIFN-γ能否抑制猪传染性胃肠炎病毒在PK-15细胞上的增长进行测定分析。结果表明：通过SDS-PAGE、Western-blot证明了重组蛋白(rPoIFN-γ)在重组杆状病毒(Bac-IFN-γ)感染的sf9昆虫细胞中得到表达，在抗病毒活性试验中显示rPoIFN-γ能一定程度抑制TGEV在PK-15细胞上的复制。说明重组猪γ-干扰素有成为治疗猪传染性胃肠炎病毒，乃至其他猪传染病的蛋白类药物的潜力。
Abstract: In this experiment, the Fc-to-Bac baculovirus/insect cell expression system was used to amplify and clone the IFN-γ gene of Rongchang porcine, and the donor plasmid pFastBacTM Dual-IFN-γ containing the completely open reading frame of Rongchang pig IFN-γ was constructed. The re-combinant plasmid was transformed into DH10Bac competent cells, and the baculovirus recom-binant shuttle plasmid Bacmid-IFN-γ was obtained, and the sf9 insect cells in the logarithmic growth phase were transfected to obtain recombinant baculovirus rBac-IFN-γ. Observing cyto-pathic effect after inoculation of TGEV on pig kidney cells (PK-15) infected with recombinant ba-culovirus and using PT-PCR technology to inhibit rPoIFN-γ on porcine transmissible gastroenteritis virus on PK-15 cells, the growth is measured and analyzed. The results showed that the recombinant protein (rPoIFN-γ) was expressed in recombinant baculovirus (Bac-IFN-γ)-infected sf9 insect cells by SDS-PAGE and Western-blot, and rPoIFN-γ was shown in antiviral activity test. It can inhibit the replication of TGEV on PK-15 cells to a certain extent. This indicates that recombinant porcine interferon-γ has the potential to be a protein drug for the treatment of porcine transmissible gastroenteritis virus and even other porcine infectious diseases.
文章引用：王凯, 杨洋, 余秋寒, 黄石磊, 谢录异, 冉玲, 宋振辉. 荣昌猪γ干扰素重组杆状病毒的构建及其抗病毒活性分析[J]. 微生物前沿, 2019, 8(1): 40-50. https://doi.org/10.12677/AMB.2019.81006

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