摘要
构建青海花斑裸鲤肝胰腺组织的cDNA文库并鉴定文库质量．提取青海花斑裸鲤肝胰腺组织总RNA，利用SMART（switching mechanism at 5’end of RNA transcript）技术，使用含有SfiIB酶切位点的oligo（dT）引物和含有SfiIA酶切住点的SMART IVTM寡核苷酸，在PowerScriptTM逆转录酶作用下，运用mRNA5’末端的模板转换方法合成cDNA第一链,利用LDPCR（long-distance PCR）扩增双链cDNA，经SfiI（IA和IB）酶切后，通过CHROMA SPIN-400柱进行分级分离去除〈500bp的片段，再同经SfiI酶切的）,Trip IEx2载体连接，体外包装后转染到大肠杆菌XL1-Blue宿主菌中，进行文库滴度和重组率的测定，然后扩增文库并随机挑取30个噬菌斑进行PCR反应鉴定插入片段大小．构建的cDNA文库滴度为1．8×10^6pfu／mL，重组率〉98％，文库扩增后滴度达8×10^9pfu／mL，插入片段长度在0．37～3．5kb之间，平均长度为1．56kb．文库具有良好的质量，为进一步筛选、克隆青海花斑裸鲤肝胰腺组织特异表达基因奠定了基础．
In order to construct the full length cDNA library of the hepatic and pancreatic tissues of Gymnocypris przewalskii and identify the quality of the library, the total RNA was extracted from the hepatic and pancreatic tissues of Gyrnnocypris przewalskii. The full length cDNA library was constructed with SMART （switching mechanism at 5＇ end of RNA transcript） technique. Titer of the unamplified library was done and the percentage of recombinant clones was determined, and at last, to amplify the library and pick thirty phage plaques randomly to identify the size of the inserts by using PCR reaction. The results showed that the titer of the constructed cDNA library was 1.8×10^6 pfu/mL in which the percentage of recombinant clones was above 〉98%, and the titer of amplified library was 8×10^9 pfu/mL. The insert size ranged from 0.37 to 3.5kb and the average length was 1.56 kb. The conclusion was that the quality of cDNA library of hepatic and pancreatic tissues of Gyrnnocypris przewalskii was excellent, which established a solid foundation for the further screening and cloning the specific expression genes.
出处
《农业科学研究》
2006年第2期15-19,共5页
Journal of Agricultural Sciences
基金
青海大学医学院基础医学部资助项目
