环形泰勒虫裂殖体感染的牛淋巴细胞中galectin-1基因的原核表达与鉴定  

Prokaryotic expression and identification of galectin-1 gene from bovine lymphocytes infected with Theileria annulata schizont

作　　者：李亚琼[1,2] 李有全[2] 韩元[1] 李倩[2] 徐春兰[1] 秦鸽鸽[1,2] 殷宏[2,3] 蔡葵蒸[1] 

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州730030 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2015年第5期497-502,共6页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31272556);农业部肉牛牦牛项目(CARS-38);农业科技创新工程项目(ASTIP);国家农业公益性行业科研专项(201303035)

摘　　要：为了获得重组牛半乳糖凝集素1(galectin-1),从被环形泰勒虫裂殖体感染的牛淋巴细胞中提取总RNA,反转录合成第一链cDNA,利用RT-PCR扩增galectin-1基因。将目的片段连接到载体pET-30a(+)上,并转化入表达菌BL21(DE3)中,经PCR、酶切及测序验证正确后,进行IPTG诱导表达和His-NiResin蛋白纯化。SDS-PAGE结果表明,获得了约17ku的表达产物。Western-blot分析表明,表达产物与抗His标签的小鼠单克隆抗体、绵羊抗牛半乳糖凝集素1多克隆抗体均能发生反应。重组半乳糖凝集素1的多克隆抗体也可以较好地识别天然蛋白。本试验成功表达了牛半乳糖凝集素1,并制备了其多克隆抗体,为以后进行与半乳糖凝集素1相互作用蛋白的研究奠定了基础。To gain recombinant bovine galectin-1,the first strand cDNA was synthesized based on the total RNA that extracted from bovine lymphocytes infected with Theileria annulata schizonts.A galectin-1gene was amplified by RT-PCR.Then the fragment was linked into the vector pET-30a(+)and transformed into Escherichia coli BL21(DE3)strain.The recombinant protein was expressed and purified after the recombinant plasmid pET-30a(+)-galectin-1was identified by PCR,restriction enzyme digestion and nucleotide sequencing.The recombinant protein was about 17 ku in size confirmed by SDS-PAGE,and Western-blot analysis indicated that the bovine galectin-1protein reacted with the mouse monoclonal antiHis tag and the sera from sheep immunized with the recombinant galectin-1.In addition,the anti-galectin-1polyclonal antibody possessed good reaction with nature Galectin-1protein.The above-mentioned results laid the foundation for studies of interacting protein with galectin-1in the future.

关 键 词：牛 半乳糖凝集素1 原核表达 多克隆抗体 

分 类 号：S852.7[农业科学—基础兽医学]