摘要
目的构建小鼠神经细胞cDNA T7噬菌体表面展示文库。方法用Trizol试剂提取原代小鼠神经细胞总RNA,分离、纯化mRNA,经反转录合成其双链cDNA。在双链cDNA末端加上定向EcoRⅠ/HindⅢ接头,用内切酶EcoRⅠ和HindⅢ消化接头,以形成两端分别带有EcoRⅠ和HindⅢ黏性末端的双链cDNA。经Mini Column进行纯化,收集大小在300 bp以上的双链cDNA片段,将其连接于含有EcoRⅠ和HindⅢ末端的T7Select 10-3b载体上,体外包装后经BLT5403菌株增殖,进行文库扩增。结果所构建的文库库容量为含有3.04×107个重组子,原始滴度为2.8×107 pfu/ml,重组率为92%,扩增后文库滴度为3.5×1010 pfu/ml。对随机挑取的100个噬菌斑进行PCR鉴定,88%的插入片段大于300 bp。结论 成功构建了小鼠神经细胞cDNA T7噬菌体表面展示文库。
Objective To construct the cDNA T7 phage surface display library of mouse neurocytes.Methods Total RNA of primary mouse neurocytes was extracted with Trizol reagent,from which mRNA was isolated and purified,and used for synthesis of double-stranded（ds） cDNA by reverse transcription.Directional EcoRⅠ / HindⅢ linker were added at the ends of ds cDNA,then digested with EcoRⅠ and HindⅢ to obtain a ds cDNA with both EcoRⅠand HindⅢ l cohesive ends.The ds cDNA fragments at lengths of more than 300 bp were collected by using Mini Column Fractionation Kit,and ligated to T7Select 10-3b vector with EcoRⅠ and HindⅢ ends,which was,after packaging in vitro,transformed to BLT5403 cells to construct the T7 phage display library.Results The constructed library,with a primary titer of 2.8 × 107 pfu / ml,contained 3.04 × 107 recombinants,of which the recombination rate and titer after amplification were 92% and 3.5 × 1010 pfu / ml respectively.A total of 100 plaques were selected randomly and identified by PCR,and the result showed that the lengths of 88% of inserted fragments were more than 300 bp.Conclusion The cDNA T7 phage surface display library of mouse neurocytes were successfully constructed.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第9期1013-1015,1021,共4页
Chinese Journal of Biologicals
基金
国家自然科学基金(31072134
30871849)
吉林省自然科学基金(201115040)
