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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 expressing both human β-defensin 3 (hBD3) and connective tissue growth factor (CTGF) in vitro and determine the transfection efficiency and expression of BMSC. Methods Recombinant lentiviral vector containing hBD3, CTGF and enhanced green fluorescent protein (EGFP) gene was constructed in vitro and the virus titer was determined. BMSCs were transfected to determine the optimal fold of infection (MOI) value. The transfection efficiency and passage stability of the virus were detected by fluorescence microscopy and flow cytometry. Western blotting was used to detect the expression of the target protein. Results The recombinant lentiviral vectors Lenti-CTGF-hBD3-EGFP, Lenti-hBD3-EGFP and Lenti-EGFP were successfully constructed by PCR and sequencing analysis. The titer of the recombinant lentivirus was 3.21 × 10 -8 , 5.80 × 10 ~ 8 and 1.16 × 10 ~ 9. The optimal MOI value of virus transfection BMSC was 150. Under fluorescence microscope, the green fluorescent efficiency of BMSC was high after transfection. The transfection efficiency of Lenti-CTGF-hBD3-EGFP was 79.72%, Lenti-hBD3-EGFP was 83.08% and Lenti-EGFP was 84.43% , And are stable genetic. Western blotting showed that the protein of interest was expressed successfully. Conclusion The recombinant lentiviral vector co-expressing hBD3 and CTGF was successfully constructed and expressed efficiently and stably in BMSC, which laid the foundation for further study on the application of hBD3 and CTGF.