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4×10^6pfu/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。
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× 10^6 pfu/mL and the length of inserts was about 100-3 000 bp. It is helpful in the further study on screening related genes.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2007年第9期999-1002,共4页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
科技部社会公益研究专项项目"畜禽重大疫病监测与控制系统研究"(2001DIA10006)
