鸡毒霉形体TM-1基因原核表达载体的构建及表达  

Expression vector construction and prokaryotic expression of TM-1 gene of Mycoplasma gallisepticum H3 strain

作　　者：郝永清[1] 王秀青[1] 周艳君[2] 童光志[2] 高金亮[3] 赵振华[1] 乌尼[1] 

机构地区：[1]内蒙古农业大学动物科学与医学学院,内蒙古呼和浩特010018 [2]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001 [3]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《中国兽医科技》2004年第4期18-20,共3页Chinese Journal of Veterinary Science and Technology

摘　　要：对从含有鸡毒霉形体H3株TM 1基因的大肠埃希氏菌DH5α中提取的质粒,进行EcoRⅠ和SalⅠ双酶切,获得了大约为786bp的目的片段。将此片段与经EcoRⅠ和SalⅠ双酶切的线性原核表达载体pET 30a 1连接,转化宿主菌BL21(DE3),筛选阳性克隆。提取质粒,经酶切、PCR扩增和测序分析确证其插入正确,从而构建成了重组表达载体。阳性克隆用IPTG诱导表达,表达产物经SDS PAGE电泳分析,证明其分子质量约为29ku;Western blotting分析表明,该蛋白可被鸡毒霉形体阳性血清识别,具有生物学活性。The abstracted recombinant plasmid of TM-1 gene of Mycoplasma gallisepticum(MG) H3 strain was digested with EcoRⅠ and SalⅠ. The obtained fragment 786 bp in length was inserted into the prokaryotic expression vector pET-30a-1 digested by the same restriction endonucleases and transformed into BL21(DE3). The insert position, size and open reading frame (ORF) were all confirmed to be right by PCR, restriction digestion and sequencing analysis. Thus the prokaryotic expression vector was (constructed) successfully. Positive clone was induced with IPTG for expression of TM-1 gene. SDS-PAGE (analysis) showed that molecular weight of the expressed protein was 29 ku. Western-blotting result showed that the recombinant protein can be recognized by positive serum of MG.

关 键 词：鸡毒霉形体 TM-1基因 原核表达 载体 生物学活性 克隆 质粒 PCR SDS—PAGE 分子质量 

分 类 号：S852.62[农业科学—基础兽医学]