恒河猴干扰素-γ基因的原核表达及其免疫活性的初步研究  

Prokaryotic expression of Macaca mulatta interferon-gamma and preliminary research of its biouctivities

作　　者：弓超[1,2] 王文博[2] 钟志军[1] 曹雪峰[1] 郭平[2] 李威[1] 邓磊[1] 冯帆[1] 张粤[1] 范泉水[2] 邱薇[2] 彭广能[1] 

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]成都军区疾病预防控制中心,四川成都610021

出　　处：《中国兽医科学》2017年第3期346-351,共6页Chinese Veterinary Science

基　　金：公益性行业(农业)科研专项(201303042);国家自然科学基金项目(31272620);国家重点研究发展计划项目(2016YFD0501009)

摘　　要：为构建以恒河猴干扰素-γ为目的基因的原核表达重组质粒并进行免疫活性分析,获得具有生物学活性重组IFN-γ蛋白。应用逆转录-聚合酶链式反应(RT-PCR)从恒河猴外周血单个核细胞(PBM C)扩增干扰素-γ基因,然后将其克隆至表达载体p ET32a(+)中,转化BL21(D E3)进行诱导表达。SDS-PAG E电泳分析其可溶性,W estern-blot检测纯化后的重组蛋白,采用淋巴细胞增殖试验检测重组IFN-γ的活性。结果显示,成功构建恒河猴干扰素-γ原核表达重组菌,重组蛋白分子质量约为35.4 ku,主要以包涵体形式存在。重组IFN-γ具有促进淋巴细胞转化增殖的生物学活性。结果表明,本试验成功获得了重组IFN-γ蛋白,表达量高且具有生物学活性,为重组IFN-γ治疗恒河猴疾病奠定了基础。Recombinant prokaryotic expression plasmid was construxted based on Macaca mulatta interferon-γ and its immuno-competence was analyzed,in order to obtain the recombinant IFN-γ protein with biological activity.A IFN-γ gene was amplified from porcine peripheral blood mononuclear cell（PBMC） by reverse transcription polymerase chain reaction（RT-PCR） and cloned into p ET32a（＋） to construct a recombinant plasmid.These recombinant plasmids were induced in E.coli BL21（ DE3） after transformation.The solubility of recombinant protein was analyzed by SDS-PAGE.The recombinant IFN-γ protein was detected by Western-blot and the biological activity of the purified protein was detected by lymphocyte proliferation assay.The recombinant prokaryotic expression plasmid was successfully constructed and highly purified protein was expressed.The molecular weight of recombinant protein was about 35.4 ku and it was expressed in the form of inclusion bodies.The recombinant IFN-γ had the ability to stimulate transformation and proliferation of lymphocyte.The recombinant IFN-γ protein of Macaca mulatta was expressed effectively and had high biological activities.This study laid the foundation for further studies on treatment of the disease caused by Macaca mulatta.

关 键 词：恒河猴 干扰素-Γ 原核表达 生物学活性 

分 类 号：S852.659.5[农业科学—基础兽医学]