Construction of a yeast two-hybrid cDNA library from second-generation merozoites of Eimeria tenella
Construction of a yeast two-hybrid cDNA library from second-generation merozoites of Eimeria tenella
复制标题
柔嫩艾美耳球虫第二代裂殖子酵母双杂交cDNA文库的构建
DOI：
10.5897/ajmr12.1930
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发表时间：
2013
影响因子：
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通讯作者：
Bing Huang
中科院分区：
文献类型：
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作者：
P. Xue;H. Han;Ya;Lianlian Jiang;C. Kong;Hui Dong;Qiping Zhao;Ye Wang;Shunhai Zhu;Ting Li;F. Shu;Bing Huang
为构建柔嫩艾美耳球虫第二代裂殖子酵母双杂交文库，采用Trizol试剂提取柔嫩艾美耳球虫EtsMZ的总RNA，并从总RNA中提取mRNAs。用MMLV逆转录合成第一链cDNA。采用远距离聚合酶链式反应(LD-PCR)获得的dscDNA，经Chroma Spin TE-400柱纯化后，以酵母表达载体pGADT7-Rec为载体，按酵母交配筛选方法转化AH109酵母感受态细胞。收集所有克隆，构建了酵母双杂交文库。结果表明，文库容量为3.22×1015CFU，滴度为8.05×1012CFU/mL。PCR扩增结果表明，文库中含有约96%的重组克隆，插入片段大小在300~2000bp之间。此外，还从构建的文库中扩增出柔嫩艾美耳球虫的5个特异基因。结果表明，成功构建了EtsMZ酵母双杂交文库。该文库为利用酵母双杂交方法筛选柔嫩艾美耳球虫裂殖子侵袭相关相互作用蛋白奠定了基础。
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关键词：球虫，柔嫩艾美耳球虫，第二代裂殖子，酵母双杂交，cDNA文库。
To construct a yeast two-hybrid cDNA library from second-generation merozoites ofEimeria tenella (EtsMZ), total RNA of EtsMZ was extracted using Trizol reagent and mRNAs were isolated from total RNA. The first-strand cDNAs were synthesized by reverse transcription using MMLV. The dscDNAs acquired by Long-distance Polymerase Chain Reaction (LD-PCR) and purified by Chroma Spin TE-400 Column were transformed into AH109 yeast competent cells with pGADT7-Rec, a yeast expression vector, according to the screening method of yeast mating. All clones were harvested and the yeast two-hybrid cDNA library of the EtsMZ was constructed. The results show that the capacity and titer of the library were 3.22×1015 cfu and 8.05×1012 cfu/mL, respectively. PCR amplification revealed that the library contained approximately 96% recombinant clones, and the inserted cDNA fragments were between 300 and 2000 bp. In addition, five specific genes of E. tenella were amplified from the constructed cDNA library. It could be concluded that a yeast two-hybrid cDNA library of the EtsMZ was successfully constructed. The library can provide a foundation for screening invasion-related interaction proteins of E. tenella merozoite by yeast two-hybrid method.
Key words: Coccidia, Eimeria tenella, second-generation merozoite, yeast two-hybrid, cDNA library.
影响因子：
5.5
作者：
Shirley, Martin W.;Smith, Adrian L.;Blake, Darner P.
通讯作者：
Blake, Darner P.