草鱼细胞系CIK酵母双杂交cDNA文库的构建及初步应用

摘要

In order to explore the interactions between grass carp reovirus and the host cell proteins, the yeast two-hybrid cDNA library from grass carp CIK(Ctenopharyngodon idellus kidney)was constructed by SMART technology. Total RNA of the CIK cells was extracted and mRNA was purified. Then, mRNA as the template, the first strand of cDNA was synthesized by reverse transcription. The double-stranded cDNA was amplified through the long-distance PCR with the DNA polymerase. The cDNA library from grass carp CIK cells was constructed in the yeast strain 187, using SMART technology and the homologous recombination method. After testing, the conversion rate and the capacity of the original library was 1.6×105 and 2.4×106, respectively. The length of the inserted double-stranded cDNA fragments were 250-2 000 bp. The titer of the library was 7×107 CFU/mL and the recombination rate was 98%. This library had the well polymorphism and the integrity of the cDNA fragments. Using the constructed yeast two-hybrid from the CIK cells, the VP7 and VP5 protein in grass carp reovirus were as the baits for the screening experiment. The positive colonies of the interacting proteins of VP7 had been acquired, without the positive colony of the interacting protein of VP5. The construction of the yeast two-hybrid cDNA library from the grass carp CIK cells provided an important research tool for the study on the interaction mechanism of grass carp reovirus with the host cell.%旨在探索草鱼呼肠孤病毒与宿主细胞蛋白质间的相互作用，运用SMART技术构建了草鱼肾组织细胞系CIK （Ctenopharyngodon idellus kidney）的酵母双杂交cDNA文库。提取CIK细胞总RNA后，分离纯化mRNA，然后以mRNA为模板，反转录合成cDNA第一链，再在DNA聚合酶作用下，通过长距离PCR，扩增双链cDNA。利用SMART技术，通过同源重组的方法，在酵母株Y187中构建了草鱼CIK细胞全长cDNA文库。经检测，未扩增文库的转化率为1.6×105，文库容量为2.4×106，插入的双链cDNA片段的长度为250-2000 bp，文库滴度为7×107 CFU/mL，重组率为98%，此文库具有良好的cDNA片段多态性和完整性。利用构建的CIK酵母双杂交文库，以草鱼呼肠孤病毒的VP7和VP5蛋白作为诱饵进行筛选试验，得到VP7相互作用蛋白的阳性菌落，未得到VP5相互作用蛋白的阳性菌落。草鱼CIK细胞酵母双杂交cDNA文库的构建为研究草鱼呼肠孤病毒与宿主细胞间的互作机制提供了重要研究工具。