马γ-干扰素成熟蛋白基因在大肠埃希氏菌中表达及其多克隆抗体制备  

Expression of mature equine interferon-gamma in Escherichia coli and preparation of antisera agaist equine interferon-γ

作　　者：白宇[1] 童铁钢[1] 刘光亮[1] 肖一红[1] 王群[1] 徐树兰[1] 张维军[1] 吴东来[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2007年第11期878-882,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金项目(ZJN-0602-01)

摘　　要：采用RT-PCR从ConA刺激的马外周血单核细胞(PBMC)中扩增获得含信号肽的马γ-干扰素(Equine interferon-γ,EIFN-γ)基因,将其克隆至载体pCR2.1-TOPO中。通过PCR方法从重组质粒(pCR-EIFN-γ)中扩增马干扰素成熟蛋白(Mature EIFN-γ,reEIFN-γ)基因,并将其亚克隆至原核表达载体pET-28a(+)。重组子经测序鉴定正确后转化至大肠杆菌BL21(DE3),经IPTG诱导,对其产物进行SDS-PAGE分析和Western blot鉴定,将纯化后的表达产物免疫新西兰白兔制备多克隆抗体。结果表明,mEIFN-γ基因全长为441bD,含一个开放阅读框,编码146个氨基酸的成熟蛋白;对其表达产物以可溶性和包涵体两种形式存在,重组蛋白相对分子量约为18Ku,且具有免疫反应活性。mEIFN-γ基因在大肠杆菌中高效表达并在非变性和变性条件下,采用Ni^+亲和层析纯化方法均可获得高纯度的重组马γ-干扰素制备的多克隆抗体,为建立马γ-干扰素检测方法、监测机体免疫状态和研究机体免疫机制奠定了基础。The equine interferon-γ（EIFN- γ） gene was amplified by RT-PCR from total RNA of the ConA stimulated equine peripheral blood mononuclear cells （PBMC） and cloned into the vector PCR2.1-TOPO. DNA fragment encoding mature EIFN-γ（mEIFN-γ） was subcloned into pET-28a （＋）. The full length of mEIFN- γ gene was 441 bp in length, and contained an open reading frame encoding a protein of 146 aa. The recombinant plasmid pET-mEIFN- γ was transformed into E.coli BL21 （DE3） and fusion protein was expressed and purified using Ni^＋ column chromatography in both non-denatured and denatured conditions, and analysed by SDS-PAGE and Western blot. Rabbits were immunized with the purified EIFN- γ to generate antisera. Results showed that the expression product was about 18 Ku and possessed good immunological activity.

关 键 词：马γ-干扰素 表达 纯化 多克隆抗体 

分 类 号：Q786[生物学—分子生物学]