小鼠巨噬细胞cDNA酵母表达文库的构建与鉴定

Objective To construct and identify the cDNA library suitable for yeast expression of mouse macrophage by Gateway technique. Methods The mRNA extracted from mouse macrophage was purified. Moreover, cDNA were synthesized by biotin-conjugated Oligo (dT) primer. The double-strand cDNA was bound to attB Adapter and then purified by the cDNA size fractionation columns. BP recombination reaction between the attB- flanked cDNAs ( ＞500 bp) and attP- containing donor vector pDONRTM222 was performed, and the products were transformed into ElectroMAXTM DH10BTMT1 Phage Resistant Cells to generate a Gateway entry cDNA library. Colonies were randomly selected from the plating assay plates. Plasmid DNA was isolated and the cDNA library qualified by plasmid digestion. A reading frame cassette to pGADT7 vector was ligated to construct a Gateway destination vector. The cDNA entry library was transformed into yeast expression library after LR recombination reaction with destination vector and the Gateway entry cDNA library. Results An entry cDNA library was constructed with a titer of (6.80±0.10)× 105 cfu/ml, total clones of 7.48×106 cfu, an average insertion size of about (2.20±0.20) kb and the percentage of recombinant clones was 100%. A yeast expression library was constructed with a titer of (3.24±0.10)× 106 cfu/ml, total clones of 3.89×107 cfu, a mean insertion size about (2.27±0.15) kb and the percentage of recombinant clones was 95.83% (23/24). Conclusion The entry cDNA library and the yeast expression cDNA library meet the requirements of standard library, thereby can be used in further study.

Construction and identification of yeast expression cDNA library of mouse macrophage