人CARABIN蛋白过表达慢病毒载体构建及其稳定表达细胞株的筛选  

Construction of lentivirus expression vector of human CARABIN and screening of cell line stably expressing CARABIN

作　　者：刘凯玉[1] 蒋维[2] 王瑞娜[2] 吴家雪[1] 党永军[2] 

机构地区：[1]复旦大学生命科学学院,上海200000 [2]复旦大学基础医学院,上海200032

出　　处：《生物学杂志》2016年第3期1-4,共4页Journal of Biology

基　　金：国家自然科学基金(21572038)

摘　　要：构建并鉴定人CARABIN蛋白过表达慢病毒载体,筛选CARABIN过表达的弥散性大B淋巴瘤细胞SUDHL-6的稳定细胞株。通过设计Carabin上下游引物,用同源重组的方法构建慢病毒载体pLENTI-c GFP-CARABIN,将其与psPAX2和pMD2.G包装质粒按3∶2∶1的比例用PEI Transfection Reagent共转染HEK293T细胞制备病毒,用病毒颗粒感染SU-DHL-6细胞2周后,用流式细胞仪分选GFP表达阳性的细胞,Western Blot检测CARABIN-GFP融合蛋白表达。成功构建pLENTI-c GFP-CARABIN慢病毒过表达载体。包装病毒颗粒并感染SU-DHL-6细胞后,荧光显微镜检测和流式细胞分选鉴定表达效率为26.5%,筛选出的GFP阳性细胞用Western Blot检测到CARABINGFP融合蛋白表达。成功构建了Carabin过表达慢病毒载体并筛选出了CARABIN-GFP稳定表达的细胞株。The aim of this study is to construct the lentivirus expression vector of human CARABIN and to screen the Diffuse Large B Cell Lymphomas（ DLBCL） cell SU-DHL-6 for stable expression of CARABIN. ORF of Carabin was amplified by PCR,then the lentiviral vector of over-expression Carabin was constructed using the method of Homologous recombination,confirmed by DNA sequencing and named pLENTI-c GFP-CARABIN. The constructed recombinant vector was co-transfected with psPAX2 and pMD2. G into HEK293 T cells to produce the lentiviral particles on the ratio of 3 ∶ 2 ∶ 1 by PEI transfection reagent. The lentiviral particles were transduced into SU-DHL-6 cells and the infection efficiency was measured by Fluorescence Microscope. The cells were sorted by the Flow Cytometer（ FCM） after 2 weeks. The expression of CARABIN and GFP in the stable cell lines was detected by Western Blot. The lentiviral vector pLENTI-c GFP-CARABIN for overexpression in blood cells was constructed and validated by sequencing. The cell line of stably overexpresiong CARABIN in SU-DHL-6 was confirmed by Western Blot. The over-expression lentiviral vector of human Carabin has been successfully constructed and cell lines for stable expression of CARABIN have been successfully established. This will shed a light on the future study of CARABIN＇s function in a variety of lymphoma or leukemia models.

关 键 词：CARABIN 过表达 稳定细胞株 弥散性大B淋巴瘤 

分 类 号：Q782[生物学—分子生物学]