白细胞免疫球蛋白样受体LILRB4条件表达小鼠模型的构建

Construction of mice with conditionally expressed leukocyte immunoglobulin-like receptor LILRB4

目的将灵长类动物lilrb4基因表达在小鼠髓系细胞表面,以构建人白细胞免疫球蛋白受体亚家族成员B4(LILRB4)免疫耐受小鼠,为LILRB4功能研究提供小鼠模型。方法利用NCBI数据库分析人lilrb4与小鼠Gp49b基因的同源性。在小鼠rose26基因位点通过同源重组的方式,定点插入含有LoxP位点的人lilrb4基因编码序列,获得Rosa26^(LSL/LSL)小鼠,与Lyz2-Cre小鼠进行杂交,在小鼠髓系细胞切除LoxP序列,进而启动下游人lilrb4基因的表达。应用PCR进行小鼠基因型鉴定,通过流式细胞仪检测Rose26^(LSL/LSL);Cre小鼠外周血以及骨髓中髓系细胞人LILRB4的水平。结果人lilrb4与小鼠Gp49b基因比对结果表明,无论是碱基还是氨基酸序列,两者都存在较大差异,无明显同源性。PCR结果表明,Rose26^(LSL/LSL);Cre小鼠能够检测到相应的目的条带,而且在髓系细胞条件表达人lilrb4基因,不影响小鼠的表型。流式细胞仪检测结果显示,Rose26^(LSL/LSL);Cre小鼠的髓系细胞特异性表达人LILRB4。结论Rose26^(LSL/LSL);Cre小鼠的髓系细胞中能够检测到人LILRB4的表达,本研究成功构建了人LILRB4耐受的基因敲入小鼠模型。

Objective To conditionally express primate Human leukocyte immunoglobulin receptor subfamily member B4(lilrb4)gene on the surface of mouse myeloid cells to construct a mouse model of human LILRB4 immune tolerance,so as to provide a mouse model for the study of LILRB4 function.Methods The homology of human and mouse lilrb4 was analyzed with NCBI database.Rosa26 LSL/LSL mice were obtained by site-directed insertion of LILRB4 gene expression frame containing LoxP site at the Rose26 gene locus by homologous recombination.Rosa26 LSL/LSL mice hybridized with Lyz2-Cre mice,excised the LoxP sequence in mouse myeloid cells,and subsequently started the downstream human LILRB4 expression.PCR was used to identify the genotypes of mice,and flow cytometry was used to detect human LILRB4 levels in peripheral blood and bone marrow of the Rosa26 LSL/LSL;Cre mice.the expression level of LILRB4 in myeloid cells in peripheral blood and.Results The results of gene and protein alignment between human lilrb4 and mouse Gp49b showed that there were significant differences in both base and amino acid sequences,without obvious homology.PCR results showed that corresponding target bands could be detected in Rose26^(LSL/LSL);Cre mice,and the human LILRB4 gene was conditionally expressed in myeloid cells without affecting the mouse phenotype.Flow cytometry showed that myeloid cells of Rose26^(LSL/LSL);Cre mice specifically expressed human LILRB4.Conclusion The expression of human LILRB4 could be detected in myeloid cells of Rosa26 LSL/LSL;Cre mice,and the gene knockin of human LILRB4 tolerance was successfully constructed in mice in this study.