鸡γ-干扰素在杆状病毒系统中的表达  

Expression of Recombinant Chicken IFN-γ in Baculovirus Vector System

作　　者：戴华[1] 季琰[1] 郑佳玉[1] 徐正中[1] 潘志明[1] 焦新安[1] 

机构地区：[1]扬州大学江苏省人兽共患病学重点实验室,江苏扬州225009

出　　处：《生物技术通讯》2009年第6期810-813,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30425031);全国优秀博士学位论文作者专项基金(FANEDD200358);国家公益性行业科研专项经费项目(200803020)

摘　　要：目的:获得具有生物学活性的重组鸡γ-干扰素(ChIFN-γ)。方法:应用RT-PCR方法扩增ChIFN-γ基因cDNA,将其克隆入杆状病毒表达系统的转移载体pFastBac1,构建重组质粒pFast-ChIFN-γ,通过位点特异性转座,将ChIFN-γ基因整合到Bacmid穿梭载体中,构建重组质粒pBac-ChIFN-γ并转染昆虫细胞Sf9,采用间接免疫荧光试验(IFA)、ELISA试验鉴定重组ChIFN-γ的表达,通过细胞病变抑制法检测重组蛋白的抗病毒活性。结果:IFA、ELISA与细胞病变抑制试验结果显示,重组ChIFN-γ在杆状病毒系统中获得表达,其抗病毒活性达5×103～1×104U/mL。结论:重组ChIFN-γ的获得为其开发应用提供了重要的生物材料。Objective： To abtain the recombinant chicken interferon gamma（ChIFN-γ） with the bioactivity in baculavirus system. Methods： ChIFN-γ gene was amplified by RT-PCR and cloned into transfer vector pFastBacl to construct recom- binant plasmid pFast-ChIFN-γ Then the recombinant plasmid was transformed into E.coli competent cells DH10Bac which contained the bacmid with a mini-att Tn7 target site and the helper plasmid. The recombinant pBac-ChIFN-γ, was gener- ated by transposing the mini-Tn7 element located in pFast-ChIFN-γ to the mini-att Tn7 attachment site on the Bacmid. Subsequently the recombinant pBac-ChIFN-γ was transfected into the Sf9 insect cells mediated by lipofectin to produce recombinant baculovirus and express recombinant ChIFN-γ products （Bac-ChIFN-γ）, which was analyzed by indirect immunofluorescent assay（IFA） and ELISA post-transfection. The biological activity of Bac-ChIFN-γ was identified by its inhi- bition to vesicular stomatitis virus-induced cytotoxicity of chicken embryonic fibroblasts in vitro. Results： IFA and anti- virus activity results showed that Bac-ChIFN-γ, was expressed successfully, and its biological activity was about 5×10^3-1×10^04 U/mL. Conclusion： The recombinant protein has provided an important biological material to develop and utilize ChIFN-γ.

关 键 词：鸡γ-干扰素 杆状病毒 重组蛋白 

分 类 号：Q78[生物学—分子生物学]