利用gateway技术构建巨型艾美耳球虫孢子化卵囊cDNA表达文库  

Construction of a cDNA expression library of Eimeria maxima sporulated oocysts using gateway technology

作　　者：李梦辉[1] 刘连瑞[1] 涂浩[1] 黄经纬[1] 张振超[1] 徐立新[1] 严若峰[1] 李祥瑞[1] 宋小凯[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2015年第4期630-635,共6页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31201896;31372428);江苏省自然科学基金项目(BK2010446);江苏高校优势学科建设工程资助项目

摘　　要：[目的]为了寻找可用于研制高效重组疫苗的抗原基因,利用gateway技术构建巨型艾美耳球虫孢子化卵囊cDNA表达文库。[方法]首先从新鲜分离的巨型艾美耳球虫孢子化卵囊中提取总RNA并分离纯化mRNA,用Clone MinerTMⅡcDNA文库构建试剂盒构建巨型艾美耳球虫孢子化卵囊cDNA初级文库,然后将cDNA从初级文库重组到p VAX1.0上,构建巨型艾美耳球虫孢子化卵囊cDNA表达文库,最后用7个已知巨型艾美耳球虫基因的引物鉴定文库。[结果]构建的巨型艾美耳球虫孢子化卵囊cDNA初级文库总容量为0.92×107CFU,插入片段平均大小为1.63 kb;表达文库总容量为2.32×107CFU,插入片段平均大小为1.64 kb。两种文库的阳性重组率均为100%。能用特异性引物从该表达文库扩增出7个已知的巨型艾美耳球虫基因。[结论]该文库质量高,代表性强,为进一步筛选免疫保护性抗原基因奠定了坚实基础。[ Objectives ] In order to find effective candidate antigen genes of recombinant vaccine, a cDNA expression library of Eimeria maxima was constructed using gateway technology in this study. [Methods]Firstly,the RNA was extracted from fresh sporulated oocysts of E. maxima and the mRNA was isolated and purified. Then the entry library of E. maxima was constructed by CloneMinerTM Ⅱ cDNA library construction kit. Secondly, the cDNA was recombined from entry library to pVAX1.0 to construct the expression library of E. maxima sporulated oocysts. Hereafter, the library was tested by PCR with the primers of known E. maxima genes. [ Results ] The total clones of the entry library was 0.92×10^7 CFU with an average inserts size of 1.63 kb,and the total clones of the expression library was 2.32×10^7 CFU with an average inserts size of 1.64 kb. The positive rates of recombination of them were 100%, and seven known E. maxima genes could be amplified using their specific primers. [ Conclusions ] The constructed library had high quality and strong representative. Our research established a solid foundation for further screening better immune protective antigen genes.

关 键 词：巨型艾美耳球虫 孢子化卵囊 GATEWAY技术 CDNA表达文库 

分 类 号：S852.73[农业科学—基础兽医学]