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

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。

In this experiment,the Fc-to-Bac baculovirus/insect cell expression system was used to amplify and clone the IFN-γgene of Rongchang porcine,and the donor plasmid pFastBacTM Dual-IFN-γcontaining the completely open reading frame of Rongchang pig IFN-γwas constructed.The re-combinant plasmid was transformed into DH10Bac competent cells,and the baculovirus recom-binant shuttle plasmid Bacmid-IFN-γwas 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-γon 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-γwas 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-γhas the potential to be a protein drug for the treatment of porcine transmissible gastroenteritis virus and even other porcine infectious diseases.