利用SMART技术构建鸡肺酵母双杂交cDNA文库

Construction of the Yeast Two-hybrid cDNA Library of Chicken Lung with SMART Technology

以1月龄SPF鸡肺为材料,用Trizol方法提取总RNA,并用mRNA纯化试剂盒纯化PolyA+mRNA,利用SMART技术合成双链cDNA(ds cDNA)。合成的ds cDNA通过CHROMA SPINTM TE-400 Column进行纯化,纯化后的ds cDNA与线性pGADT7-Rec共转化酵母感受态细胞AH109中,以同源重组的方式,在酵母细胞内构建成鸡肺的cDNA文库。获得的文库容量为3.9×106cfu,随机选取20个克隆进行PCR检测,插入片段大小集中在0.3～3.0kb之间,平均插入片段约为1.4kb左右,文库重组率为100%。结果表明该文库达到了高质量文库所应具备的条件,为酵母双杂交技术筛选与IBV-N相互作用的宿主细胞蛋白奠定基础。

The mRNA of one-month-old SPF chicken lung was purified by mRNA purification kit, and double-stranded cDNA （ ds cDNA） was synthesized using SMART technology. The ds cDNA purified by CHROMA SPINTM TE-400 Column and the linearized pGADT7-Rec vector were co-transformed into competent cells of yeast strain AH109. A chicken lung yeast two-hybrid cDNA library was constructed in yeast cells by homologous recombination. The library capacity was 3.9 ~ l06 cfu with insert size ranging from O. 3 kb to 3.0 kb, and the average length of inserts was approximately 1.4 kb with the recombination rate of 100%. These results showed that a high-quality cDNA library of chicken lung could be constructed, which could be used for studying host cell protein interaction with the N protein of Infectious Bronchitis Virus （IBV）.