DT40细胞sIgMλ轻链基因敲除载体的构建  

Construction of Knockout Vector for sIgM λ Light Chain Gene from DT40 Cells

作　　者：游雷鸣[1,2] 罗俊[1] 王爱萍[2] 张改平[1] 卜丹[2] 郭亚男[2] 祈艳华[2] 

机构地区：[1]河南省农业科学院,河南省动物免疫学重点实验室,河南郑州450002 [2]郑州大学生物工程系,河南郑州450001

出　　处：《华北农学报》2009年第3期1-6,共6页Acta Agriculturae Boreali-Sinica

基　　金：“十一五”国家科技支撑计划(2006BAD06A04-6);河南省基础与前沿技术研究计划(082300433201)

摘　　要：从鸡B淋巴DT40细胞系中克隆-βactin启动子,替换pCDNA3.1(+)载体中的SV40启动子,构建-βactin启动子驱动的Neomycin抗性基因表达框。将克隆的sIgMλ轻链基因两侧各约2 kb的序列插入到抗性表达框的两侧作为同源臂。PCR、酶切以及测序结果表明成功构建了靶向sIgMλ轻链基因的置换型打靶载体pCDNA-act-neo-HR。为建立sIgMλ轻链基因敲除的DT40细胞模型,探究sIgMλ轻链在IBDV感染DT40细胞过程中的作用奠定了基础。β-actin promotor was cloned from chicken B lympha DT40 cells and used to replace the SV40 promotor of pCDNA3.1 （ ＋ ）vector, so as to construct the Neomycin resistance express cassette driven by β-actin promotor. Then about 2 kb DNA sequences on each side of the locus of slgM λ gene were inserted into the MCS of the constructed cassette, acting as the homologous arm. PCR identification, restriction enzyme digestion and sequence analysis confirmed that the targeting replacement vector pCDNA-act-neo-HR for slgM λ gene was successfully constructed. This laid the foundation for establishing the model cell lack of slgM λ gene, and further investigating the roles of slgM λ light chain in the infection of IBDV to DT40 cells.

关 键 词：sIgMλ轻链 -βactin启动子 基因敲除 同源重组 

分 类 号：Q782[生物学—分子生物学]