人BAFF转基因斑马鱼的构建及早期免疫基因表达检测

目的建立人BAFF转基因斑马鱼模型，探讨其在自身免疫性疾病发病中的作用。方法RT-PCR法由人淋巴瘤细胞克隆了人BAFF基因全长855bp蛋白编码区域，构建表达人BAFF重组质粒Tol2-hBAFF，体外细胞转染并通过免疫印迹法验证蛋白表达。重组载体经显微注射斑马鱼受精卵后，GFP荧光跟踪并筛选阳性鱼。qPCR法检测早期免疫相关基因表达情况。结果人BAFF-GFP融合蛋白可成功表达，利用Tol2-hBAFF重组质粒显微注射斑马鱼受精卵可获得表达人BAFF的转基因斑马鱼，且表达人BAFF斑马鱼1dpf胚胎中TCRAC明显高表达，而Ikaros则表达量显著降低，表明在斑马鱼胚胎中表达人BAFF蛋白会造成早期淋巴系统中基因的过早表达。结论建立的表达人BAFF的转基因斑马鱼，可为系统性红斑狼疮等与BAFF功能亢进密切相关的自身免疫性疾病的治疗，及相关机制研究提供一种具有诸多优点的新型工具。

Construction of a transgenic zebrafish model expressing human BAFF and early detection of immune-related gene expressions

Objective To construct a transgenic zebraifsh model expressing human BAFF, and study the pathogenesis of autoimmune diseases. Methods 855 bp BAFF cDNA was cloned by RT-PCR from human lymphoma cell line to construct the BAFF expression recombinant plasmid Tol2-hBAFF. The protein expression in HeLa cells was detected by Western blot after in vitro transfection. Zebrafish embryos were injected with recombinant BAFF plasmids and screened by GFP tracing. GFP-positive embryos were selected to analyze the early expression of immune-related genes by qPCR. Results BAFF-GFP fusion protein expression was successfully carried out, which proved to be an effective method to produce transgenic zebrafish for future study, qPCR results demonstrated that TCRAC was significantly higher expressed in 1- dpf transgenic embryos while Ikaros gene was down-regulated, suggesting that BAFF overexpression in zebrafish embryo may resluh in immuno-related gene premature expression in the early lymphatic system. Conclusions The results of this study indicate that transgenic zebrafish can be obtained by microinjection of Tol2-hBAFF recombinant plasmid into zebrafish embryo. This model may provide an novel effective tool to explore the mechanisms of human systemic lupus erythematosus and its treatment.