人β-防御素3与结缔组织生长因子共表达重组慢病毒载体的构建及表达  

Construction and identification of recombinant lentivirus carrying human beta defensins-3 and connective tissue growth factor gene

作　　者：孙洁[1] 钱智勇[2] 刘静[1] 张欣然[1] 刘翠[1] 刘东华[2] 郭红延[1] 郭希民[2] 

机构地区：[1]北京武警总医院口腔医学中心,北京100039 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《军事医学》2017年第1期38-42,共5页Military Medical Sciences

摘　　要：目的体外构建共表达人β-防御素3(hBD3)与结缔组织生长因子(CTGF)的重组慢病毒,并检测其对骨髓间充质干细胞(BMSC)的转染效率及表达情况。方法体外构建携带hBD3、CTGF和增强绿色荧光蛋白(EGFP)基因的重组慢病毒表达载体,测定病毒滴度。转染BMSC,确定最适感染倍数(MOI)值。采用荧光显微镜和流式细胞仪检测病毒的转染效率及传代稳定性。Western印迹检测目的蛋白的表达。结果经PCR和测序分析鉴定,成功构建了携带hBD3和CTGF基因的重组慢病毒载体Lenti-CTGF-hBD3-EGFP、Lenti-hBD3-EGFP和Lenti-EGFP,检测病毒滴度分别为3.21×10~8、5.80×10~8和1.16×10~9。病毒转染BMSC最适MOI值为150。荧光显微镜下,转染后BMSC绿色荧光效率高;流式细胞仪检测病毒的转染效率,Lenti-CTGF-hBD3-EGFP为79.72%,Lenti-hBD3-EGFP为83.08%,Lenti-EGFP为84.43%,且都稳定遗传。Western印迹显示目的蛋白表达成功。结论成功构建了共表达hBD3、CTGF的重组慢病毒载体,并能在BMSC中高效、稳定表达,为进一步研究hBD3与CTGF的应用奠定了基础。Objective To construct a recombinant lentivirus containing human beta defensins-3（hBD3）,connective tissue growth factor gene（CTGF） and enhanced green fluorescent protein（EGFP）,and to detect its translation in rabbit bone marrow mesenchymal stem cells（BMSC）. Methods The lentivirus containing hBD3,CTGF and EGFP genes was constructed in vitro. The titer of lentivirus was tested with end-paint dilution assay. Rabbit BMSCs were transfected with recombinant virus. The best value of multiplicity of infection（MOI） was tested. The expression condition,transfection efficacy and genetic stability of the target genes were evaluated by using fluorescence microscopy and flow cytometry.Western blotting was used to detect the expression of the target protein. Results Recombinant lentivirus vectors： LentiCTGF-hBD3-EGFP,Lenti-hBD3-EGFP, and Lenti-EGFP, were successfully obtained. The titer of the recombinant lentiviruses was 3. 21 × 10^8,5. 80 × 10^8,and 1. 16 × 10^9,respectively. The best MOI value to transfect BMSCs was 150.The transfection efficacy of these lentivirus vectors was high,reaching 79. 72% as assessed by flow cytometry,and it could be stably inherited. Western blotting displayed that target protein expression was successful. Conclusion The construction of recombinant lentiviruses carrying hBD3 and CTGF genes is successful and can be effectively transfected into BMSCs.

关 键 词：重组慢病毒 人β-防御素3 结缔组织生长因子 骨髓间充质干细胞 

分 类 号：R781[医药卫生—口腔医学]