吕氏泰勒虫cDNA表达文库的构建及其抗原基因的免疫筛选  

Construction of cDNA Expression Library of Theileria luwenshuni and Immunoscreening of Antigenic Genes

作　　者：李有全[1] 罗建勋[1] 高金亮[1] 关贵全[1] 马米玲[1] 刘志杰[1] 刘军龙[1] 刘爱红[1] 任巧云[1] 党志胜[1] 鲁炳义[1] 刘光远[1] 白启[1] 殷宏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所/家畜疫病病原生物国家重点实验室/甘肃省动物寄生虫病重点实验室,兰州730046

出　　处：《中国农业科学》2009年第1期331-339,共9页Scientia Agricultura Sinica

基　　金：国家自然资源平台项目(2005DKA21100);国家自然科学基金项目(30571397;30800820);国家"863"项目(2006AA10A207);支撑计划(2007BAD40B00);国家重点试验室自主申请项目(SKLVEB2008ZZKT019);中央级公益性科研院所基本科研业务项目(BRF080303);甘肃省重大专项(0801NKDA033);中国欧盟合作项目专项

摘　　要：【目的】构建吕氏泰勒虫裂殖子cDNA表达文库,并从中筛选抗原候选基因。【方法】从吕氏泰勒虫裂殖子直接提取和纯化mRNA,采用oligo(dT)引物合成双链cDNA,并在其两端加EcoRⅠ/HindⅢ定向接头。将所产生的cDNA分子定向克隆到具有EcoRⅠ/HindⅢ粘性末端的λSCREEN载体的两臂之间。用PhageMaker extract对连接产物进行体外包装以形成完整的噬菌体,并用之转染大肠杆菌ER1647,从而构建成吕氏泰勒虫的cDNA表达文库。用吕氏泰勒虫阳性血清和兔抗绵羊IgG-AP筛选得到阳性克隆,经测序和Blast软件分析并获得新基因。【结果】成功构建吕氏泰勒虫裂殖子cDNA表达文库,其初级库容量约为1.0×106PFU,扩增文库的滴度为8.2×108PFU·ml-1,文库重组率为100%;通过免疫学筛选、测序和Blast软件分析,共获得30个新基因,其中15个基因已登录GenBank/NCBI。【结论】为研究泰勒虫疫苗、新型医药和诊断抗原,以及发展可持续性防制羊泰勒虫病提供基本材料。[objective] A cDNA expression library of the merozoites of Theileria luwenshuni was successfully constructed and antigenic genes were obtained by immunoscreening from it. [Method] Purified mRNA was isolated from the merozoites of Theileria luwenshuni. A library of oligo （dT） -primed cDNA with added directional EcoR I IHindlII linkers was constructed and ligated to the EcoR I/Hind III arms of the screen vector. The recombinant phage DNA was packaged by using PhageMaker packaging extracts, resulting in a primary cDNA library. The positive clones were immunoscreened with the positive sera against Theileria luwenshuni and the rabbit anti-sheep IgG-AP from the library. The new genes were obtained by Blast anlysis and sequencing. [Result] A cDNA expression library of the merozoites of Theileria luwenshuni was successfully constructed, with a size of 1.0 ×10^6 PFU, amplified cDNA library with a phage titer of 8.2 ×10^8 PFU·ml^-1 , and a recombinant rate of 100%. Thirty new genes were obtained by analysis of immunoscreening, sequencing and Blast, and 15 of them have been submitted in GenBank/NCBI. [ Conclusion] The experiment has established an important material bases for the development of Theileria vaccine, new pattern medicine, diagnostic antigen and so on.

关 键 词：吕氏泰勒虫 CDNA表达文库 构建 免疫筛选 

分 类 号：S852.7[农业科学—基础兽医学]