关键词: 杀菌/渗透性增强蛋白(Bpi), 条件基因打靶载体, 同源重组, Cre/LoxP系统

Abstract: Objective To construct Bpi conditional gene knockout vector will lay the foundation of preparing the Bpi conditional knockout mouse for further Bpi gene functional study.Methods Using Cre /LoxP system,two LoxP sequences were inserted into intron 1 and 3 to flox exon 2 and 3 of Bpi.The 3.1 kb Bpi genomic DNA fragment containing exon 2 and 3 used as upstream homologous arm and middle homologous arm and the 4.9 kb Bpi genomic DNA fragment used as downstream homologous arm were obtained separately by LA-PCR using mouse ES cells genomic DNA as template.The recombinant plasmid of pBSKⅡ-5SLoxP and ploxPFRTNeo-3L were then used to obtain Bpi conditional gene targeting vector by sub-cloning.Results The recombinant plasmid and final conditional targeting vector were confirmed by restriction enzyme digestion and sequencing analysis.Conclusion A conditional gene targeting vector of mouse Bpi has been successfully constructed.

Key words: Bactericidal/Permeability-increasing Protein(Bpi), Conditional knockout vector, Homologous recombination, Cre /LoxP