Construction and characterization of a cDNA library from Beijing fatty chicken liver tissue and ezpr

• 作 者: Chang-qing Liu Chun-ling Tong Xiao-na Xiu Tao-feng Lu Wei-jun Guan Yue-hui Mia
• 机 构: Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100094,China;Bioscience
• 出 处: 第十一次全国畜禽遗传标记研讨会
• 发表日期: 2008年6期
• 关键词: 基因表达 基因文库 肝组织 北京油鸡

A cDNA library from Beijing fatty chicken liver tissue was constructed according to the protocol of SMART TM cDNA library Construction Kit. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. The titers of unamplifed and amplified libraries were 1.96×106 pfu/ml and 1.64×109 pfu/ml respectively. The percentages of recombinants from both libraries were 97.8％ in unamplified library and 95.6％ in amplified library. The library was converted into pTriplEx2 plasmids in E. coli BM25.8 strain. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29％, and 0.5-1.0 kb in 35.71％。 Full length purH cDNA was inserted into multiple cloning sites between EcoR I and BamH I sites in the fusion expression vectors pEGFP-N3, pEYFP-Nl and pDsRedl-Nl, and the recombinant eukaryotic expression vectors pEGFP-N3-purH, pEYFP-Nl-purH and pDsRedl-Nl-purH were constructed with GFP as reporter gene to transfect into Beijing fatty chicken fibroblasts. The results showed that 24> 48 and 72h after transfer, the efficiencies of expression of the three recombinant fusion gene types were between 10.3％ and 53.2％, and fluorescence was distributed throughout the cytoplasm and nucleus except in the cryptomere vesicle. A high quality cDNA library from Beijing fatty chicken was successfully constructed and provided a useful resource for the functional genomic research of Beijing fatty chicken.