【摘要】 通过RT-PCR法由斑马鱼脾脏克隆B细胞刺激因子baff基因,构建过表达斑马鱼baff且携带有绿色荧光标记蛋白的重组质粒pIRES2-GFP-baff;胚胎显微注射获得转基因斑马鱼胚胎;通过GFP荧光标记跟踪并筛选转基因阳性鱼;Western blot法鉴定Baff-GFP融合蛋白表达情况;qPCR检测baff,GFP及baff下游相关基因bcl-2,il-4mR-NA表达情况。结果表明细胞、胚胎及幼鱼baff和GFP均高表达,baff下游基因bcl-2激活和il-4基因抑制表达。通过胚胎显微注射法可成功获得过表达baff的转基因斑马鱼,此研究为建立红斑狼疮转基因斑马鱼模型及高通量筛选Baff拮抗剂奠定了基础。更多还原

【Abstract】 baff full length cDNA was cloned by RT-PCR and constructed to overexpression plasmid pIRES2-GFP. The baff overexpression zebrafish were successfully obtained by microinjection and fluorescence screening. The expression levels of baff, GFP as well as baff downstream regulated genes bcl-2 and il-4 in transgenic embryos were determined by qPCR. The Western blot result confirmed the expression of Baff-GFP fused protein. The result of qPCR demonstrated the overexpression of GFP and baff in transgenic embryos and juvenile fish. However, although the expression of baff downstream regulated gene bcl-2 was significantly increased, the il-4 gene showed a impaired expression. In conclusion, the transgenic zebrafish overexpressing baff can be obtained by embryo microinjection, and this is a fundamental work for the establishment of lupus erythematosus transgenic zebrafish model and high throughput screening for Baff antagonist.更多还原