猪γ干扰素的表达及抗病毒活性研究  

The Prokaryotic Expression and Its Antiviral Activity of Porcine Interferon-γ

作　　者：梁望旺[1,2] 周丹娜[1] 杨克礼[1] 刘泽文[1] 段正赢[1] 邓均华[1] 郭锐[1] 袁芳艳[1] 徐涤平[1] 田永祥[1] 

机构地区：[1]湖北省农业科学院畜牧兽医研究所,武汉430064 [2]动物胚胎工程及分子育种湖北省重点实验室,武汉430064

出　　处：《湖北农业科学》2011年第11期2283-2286,共4页Hubei Agricultural Sciences

基　　金：国家科技支撑计划项目(2006BAD06A18);湖北省农业科技创新中心资助项目(2007-620-001-03);动物胚胎工程及分子育种湖北省重点实验室开放课题(2010ZD151)

摘　　要：利用RT-PCR技术从有丝分裂原刀豆素(ConA)诱导的长白猪外周血淋巴细胞总RNA中扩增出猪γ干扰素的成熟肽基因(mPoIFN-γ),并将其亚克隆到原核表达载体pET-28a中,构建重组表达载体pET-mPoIFN-γ。经PCR、酶切及测序鉴定后,转化到大肠杆菌BL21(DE3)进行IPTG诱导表达,经SDS-PAGE和Western-blot检测,表明猪γ干扰素获得了高效表达,并具有免疫活性,表达产物约为20.5 kDa,主要以包涵体形式存在,产物经纯化、复性得到具有生物学活性的蛋白。利用细胞病变抑制法对其在H-PRRSV/Marc-145系统上进行抗病毒活性测定,结果表明其抗H-PRRSV活性为7.68×103 U/mg。为进一步研究猪γ干扰素功能奠定了基础和理论依据。For constructing recombinant expression vector pET-mPoIFN-γ,the mature peptide gene for porcine interferon-γ（mPoIFN-γ） was amplified by RT-PCR depending on the template of total RNA which was isolated from the peripheral blood lymphocyte of ConA-stimulated Landrace swine,and then subcloned into pET-28a vector.After detection by PCR,restriction enzyme digestion and sequencing,the recombinant plasmid was transformed into Escherichia coli BL21（DE3） and inducted by IPTG.The SDS-PAGE analysis and Western blot results showed that the porcine interferon-γ was highly expressed with immune activity and the expressed product was about 20.5kDa,was existed mainly in inclusion body.The recombinant product was purified and renaturalized from inclusion body to obtain the protein with biological activity.Antiviral activity assay for PoIFN-γ was performed and evaluated by standard procedures in H-PRRSV/Marc-145（virus/cell） test system.The results showed that the titer of PoIFN-γ against H-PRRSV was 7.68×103 U/mg.The research laid a foundation for the function investigation of PoIFN-γ.

关 键 词：猪Γ干扰素 克隆 原核表达 

分 类 号：S858[农业科学—临床兽医学] Q78[农业科学—兽医学]