猪囊尾蚴半胱氨酸蛋白酶TsCL-1基因在毕赤酵母中的表达  

Expression of cysteine protease gene TsCL-1 of Cysticercus cellulosae in Pichia pastoris

作　　者：刘琼[1] 骆学农[1] 郭爱疆[1] 郑亚东[1] 潘晓梅[1] 岳城[2] 才学鹏[1] 

机构地区：[1]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 [2]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《中国兽医科学》2008年第8期680-684,共5页Chinese Veterinary Science

基　　金：国家"十一五"高技术研究发展计划(863)项目(2006AA10A207);中国农业科学院基本科研业务费专项资金项目(BRF070308)

摘　　要：采用RT-PCR方法从猪囊尾蚴总RNA中扩增出TsCL-1基因并构建了真核表达载体pPIC9K-TsCL-1,pPIC9K-TsCL-1电转化毕赤酵母GS115。采用G418抗性梯度筛选多拷贝重组菌株,经甲醇诱导表达,应用SDS-PAGE和Western-blot分析重组蛋白的表达情况。结果显示,TsCL-1与GenBank登录的TsCL的核苷酸和氨基酸的同源性分别为99.8%和100%,与肝片吸虫和大片吸虫组织蛋白酶氨基酸的同源性为76.3%。TsCL-1基因在毕赤酵母中成功表达,表达产物的分子质量为42 ku左右;Western-blot分析表明,表达产物具有反应原性。The cysteine protease TsCL-1 gene of Cysticercus cellulosae was amplified from the total RNA of C. cellulosae by RT-PCR. Then the fusion expression vector pPIC9K-TsCL-1 was constructed by inserting the amplified TsCL-1 gene into pPIC9K, and was transformed to GS115 by electroporation. Multicopy recombinant strains were screened by G418 and induced by methanol. SDS-PAGE was used to analyze expression product. The results showed that the gene shared 99.8% and 100% identity at the nucleotide and amino acid levels to the ones available in GenBank, and 76.3% amino acid similarity to cathepsin of Fasciola hepatica and Fasciola gigantica, respectively. The recombinant pTsCL-1 was expressed success- fully in Pichia pastoris, and was approximately 42 ku in molecular mass. Western-blot analysis showed that the expressed recombinant protein had reactogenicity.

关 键 词：猪囊尾蚴 半胱氨酸蛋白酶 毕赤酵母 

分 类 号：S852.734[农业科学—基础兽医学] Q786[农业科学—兽医学]