堆形艾美耳球虫孢子化卵囊cDNA文库的构建及鉴定  

Construction and Identification of cDNA Library of Eimeria acervulina Sporulated Oocysts

作　　者：闫晓菲[1] 韩红玉[2] 岳城[1] 黄兵[2] 赵其平[2] 姜连连[2] 董辉[2] 卞庆松[2] 张建哲[2] 

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室,上海200232

出　　处：《复旦学报（自然科学版）》2007年第6期937-940,946,共5页Journal of Fudan University：Natural Science

基　　金：国家科技基础条件平台建设项目(2005DKA21205-4);国家高技术研究发展计划资助项目(2006AA10A207-1)

摘　　要：构建堆形艾美耳球虫（Eimeria acervulina）孢子化卵囊cDNA表达文库,以筛选其功能性基因.用TRI-ZOL Reagent试剂提取堆形艾美耳球虫总RNA,再用Oligo（dT）12-纤维素柱从总RNA中分离mRNA,以mRNA为模板,RT-PCR法反转录合成cDNA第一链,用LD-PCR法扩增合成双链cDNA,经蛋白酶K消化、SfiⅠ酶切、CHROMA SPIN-400柱分离去除小于400 bp的片段后,将cDNA与已经SfiⅠ酶切的λTriplEx2载体按一定比例连接,经体外包装,建立堆形艾美耳球虫孢子化卵囊的噬菌体表达文库.随后测定文库容量为4.6×10^6pfu/mL,扩增文库的滴度为4.4×10^10pfu/mL,重组率达到98%,插入片段大小为750-1 000 bp,并从扩增文库中扩增出了堆形艾美耳球虫巨噬细胞游走抑制因子基因片段.The express eDNA library of Eimeria acervulina sporulated oocysts was constructed to screen its functioning gene. Total RNA was isolated from E. aceruulina using TRIZOL reagent, First-stranded eDNA was synthesized by RTPCR method, and double-stranded eDNA was synthesized using LD-PCR method. The ds-eDNAs was digested by proteinase K and Sfi I restriction enzyme, less than 400 bp fragments were .separated by CHROMA SPIN-400 column to ensure the length of inserts, then the eDNA was ligated to the Sfi I digested ）.TriplEx2 vector. After packaging, the eDNA library was constructed. The titer of primary eDNA library was 4.6 × 10^6 pfu/mL, the titer of amplified eDNA library was 4.4 × 10^10 pfu/mL, the recombination rate was 98 %, the average insert fragments was 750 - 1 000 bp, and the E. acervulina macrophage migration inhibitory factor （MIF） gene was amplified from the constructed cDNA library.

关 键 词：鸡 球虫 堆形艾美耳球虫 孢子化卵囊 CDNA文库 

分 类 号：S852.723[农业科学—基础兽医学]