藏獒犬IFNγ基因的克隆表达及其生物学活性测定  

Prokaryotic Expression and Analysis of Bioactivity of Zangao Canine Interferon Gamma

作　　者：宋世斌[1,2] 陈国华[3] 胡永浩[1] 贾怀杰[3] 何小兵[3] 景志忠[3] 仝保全[4] SONG Shi-bingl,;CHEN Guo-hua;HU Yong-hao;JIA Huai-jie;HE Xiao-bing;JING Zhi-zhong;TONG Bao-quan(College of Veterinary Medicine,Gansu Agricultural University,lanzhu,gansu 730070;State Key Laboratory on Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences;Gansu Agriculture Technology College;Lanzhou Native Mastiff Park)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]甘肃农业职业技术学院 [3]中国农业科学院兰州兽医研究所家畜与病原生物学国家重点实验室 [4]兰州原生獒园

出　　处：《畜牧兽医杂志》2018年第5期1-4,7,共5页Journal of Animal Science and Veterinary Medicine

基　　金：甘肃省高等学校科研项目资助(2015A-195)

摘　　要：通过RT-PCR技术从刺激的藏獒犬外周血淋巴细胞中成功克隆IFNγ全基因,其大小为501bp,编码167个氨基酸,前23个AA为信号肽,与GenBank上发表的犬IFNγ((NM001003174.1)比较,序列的同源性为99.4%。构建了原核表达载体pET-30a-IFNγ,转化BL2L,IPTG诱导后表达约23Kd的重组蛋白,经镍琼脂糖凝胶层析柱纯化后,纯度达90%以上;利用抑制病毒噬斑法测定重组藏獒犬γ干扰素的生物学活性,结果显示纯化的蛋白具有一定的抗病毒活性,本研究结果为进一步研究犬γ干扰素的抗病毒活性奠定基础。The Canine IFNγgene was amplified by RT-PCR from the total RNA of Canine peripheral blood leucocyte induced with LPS and Con A.The cloned Canine IFNγgene was 501 bp in length encoding apolypetide of 167 anino acid.Compared with the gene of canine（（NM001003174.1）GenBank,the nucleotide homology showed 99.4%.Then the gene was subcloned into pET-30 avector,and the recombinant plasmid named pET-30 a-IFN-γwas constructed.The express product was analyzed by SDS-PAGE and Western-blotting.Expressed IFN-γprotein was purified by Ni agarose gel.The biological characteristics of recombinant Zangao Canine IFN-γprotein have been experimented by inhibition of virus plaque assay,we have successfully cloned and express the gene of Zangao Canine IFN-γ,and purified product was verified to be inhibite the cytopathic effect,this made a basis for study the bioactivity of IFN-γ.

关 键 词：藏獒犬 IFNΓ 原核表达 活性测定 

分 类 号：S813.1[农业科学—畜牧学]