EXPRESSION OF HUMAN BETA-DEFENSIN 3 IN COS-7 CELL

To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line.

山羊乳腺上皮细胞基因转染条件的优化

为了优化外源基因转入乳腺上皮细胞的条件,本研究采用组织块培养法,从山羊乳腺组织中分离并获得了纯化的乳腺上皮细胞,通过染色角蛋白和测定生长曲线对其进行了鉴定和检测。然后利用电穿孔法将PEGFPC1载体导入乳腺上皮细胞,分别比较了在不同电压(120、140、160、180、200V)、不同电击时长(5、10、15、20ms)以及2株细胞(GMEC1、GMEC2)的不同代数(第1代、第3代、第5代、第9代)条件下的转染效率,并利用优化条件对山羊乳腺上皮细胞进行人β-防御素3基因转染,经过G418筛选,最终获得单克隆细胞,同时对获得的单克隆阳性细胞进行体外诱导表达,并对诱导产物进行Western blotting鉴定。结果表明,不同遗传背景的细胞对转染效率的影响较小,而第1代培养的细胞转染效率要显著高于传代细胞,乳腺上皮细胞在电压180V、电击时长15ms、电击1次的条件下转染效率最高,优化条件下转染、筛选和扩增后获得稳定表达人β-防御素3蛋白的单克隆乳腺上皮细胞。研究工作为乳蛋白基因表达调控机制的研究及乳腺特异性表达载体的检测提供参考资料。