军事医学2017，Vol.41Issue(1)：38-42,5.DOI:10.7644/j.issn.1674-9960.2017.01.009
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
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
作者信息
- 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
- 折叠
摘要
Abstract
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: Lenti-CTGF-hBD3-EGFP, Lenti-hBD3-EGFP, and Lenti-EGFP, were successfully obtained . The titer of the recombinant lentiviruses was 3.21 ×108, 5.80 ×108, and 1.16 ×109, 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Key words
recombinant lentivirus/human beta defensins-3/connective tissue growth factor/bone marrow mesenchymal stem cells分类
生物科学引用本文复制引用
孙洁,钱智勇,刘静,张欣然,刘翠,刘东华,郭红延,郭希民..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[J].军事医学,2017,41(1):38-42,5.
