Construction of cDNA T7 Phage Surface Display Library of Mouse Neurocytes

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.