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当前位置: 首页 > 文章 > 柔嫩艾美耳球虫子孢子cDNA表达文库的构建 畜牧兽医学报 2007,38 (9) 999-1002
Position: Home > Articles > Construction of cDNA Expression Library of Eimeria tenella Sporozoites Chinese Journal of Animal and Veterinary Sciences 2007,38 (9) 999-1002

柔嫩艾美耳球虫子孢子cDNA表达文库的构建

作  者：
林青;才学鹏;窦永喜;翟军军;闫鸿斌;张彦明;田广孚;景志忠
单  位：
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甘肃省动物寄生虫病重点实验室;西北农林科技大学动物科技学院
关键词：
柔嫩艾美耳球虫;孢子化卵囊;子孢子;cDNA表达文库;构建
摘  要：
在无RNase污染的环境下,提取柔嫩艾美耳球虫(Eimeria tenella)子孢子RNA,进而纯化mRNA,采用Oligo(dT)引物反转录合成cDNA第一链和第二链,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黏性末端的λSCREEN载体的两臂之间。用Phage Maker extract对以上连接产物进行体外包装,形成完整的噬菌体,并用该噬菌体转染大肠杆菌ER1647,进行文库容量测定和扩增。以扩增文库的DNA为模板,利用已知基因引物克隆E. tenella3-1E基因,并进行测序。结果表明,成功构建了E. tenella孢子化卵囊子孢子的cDNA文库,文库原始库容量约为4×106pfu/mL,插入片段约100~3000 bp,扩增得到特定的E. tenella3-1E基因,说明文库质量高、代表性强,为进一步从文库中筛选相关基因提供了有效的工具。
译  名：
Construction of cDNA Expression Library of Eimeria tenella Sporozoites
作  者：
LIN Qing1,2,CAI Xue-peng2,DOU Yong-xi2,ZHAI Jun-jun1,2,YAN Hong-bin2,ZHANG Yan-ming1,TIAN Guang-fu2,JING Zhi-zhong2(1.College of Animal Science and Technology,Northwest A & F University,Yangling 712100,China;2.Key Laboratory of Veterinary Parasitology of Gansu Province,State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)
关键词：
Eimeria tenella;sporulated oocysts;sporozoites;cDNA expression library;construction
摘  要：
Total RNA and subsequent mRNA were isolated from E.tenella sporozoites.A library of Oligo(dT)-primed cDNA with added directional EcoR I /Hind Ⅲ 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 library was proved to be a good quality by the control tests.The cloning efficiency was evaluated and the length of the cDNA fragment was assayed by PCR.Using the amplified library as template DNA,a pair of primers were designed according to the sequence of the E.tenella 3-1E,then the gene was amplified by PCR.The results showed that the cDNA expression library of E.tenella sporozoites was constructed and the 3-1E gene was amplified successfully.The capacity of the cDNA library was 4×106 pfu/mL and the length of inserts was about 100-3 000 bp.It is helpful in the further study on screening related genes.

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