过表达Baff转基因斑马鱼的构建

The Construction of Transgenic Zebrafish Overexpressing Baff

目的体外克隆斑马鱼baff基因并分别构建pEGFP-C1-baff、pEGFP-N1-baff、pIRES2-EGFP-baff重组质粒,通过胚胎显微注射获得过表达baff的转基因斑马鱼,以探讨其作为人类SLE模型和用于SLE药物筛选的意义。方法通过RT-PCR法由斑马鱼脾脏克隆出斑马鱼baff基因全长807 bp蛋白编码区域,分别构建baff过表达载体pEGFP-C1-baff,pEGFP-N1-baff及pIRES2-EGFP-baff重组质粒,体外细胞转染并通过免疫印迹法验证蛋白表达后,通过胚胎显微注射过表达载体,GFP荧光跟踪并筛选阳性鱼。结果通过体外细胞转染实验与免疫印迹法验证了pEGFP-C1-baff,pEGFP-N1-baff转染后细胞Baff-GFP融合蛋白的成功表达,通过胚胎显微注射与GFP荧光筛选,成功获得过表达baff的转基因斑马鱼。结论本研究所构建pEGFP-C1-baff、pEGFP-N1-baff、pIRES2-EGFP-baff重组质粒均可通过显微注射获得过表达baff的阳性转基因斑马鱼,为进一步探讨其作为人类SLE疾病模型的意义及Baff拮抗药物的筛选奠定基础。

Objective To construct pEGFP-Cl-baff, pEGFP-NI-baff, and pIRES2-EGFP-baff recombinant plasmids, and establish a SLE disease model of Baff overexpressed zebrafish by microinjection and fluorescence screening. The aim is to evaluate the meaning of Baff overexpression zebrafish in study of SLE mechanism and drug selection. Methods We cloned baff full length cDNA encoded by 807 bp nuclear acid using spleens RNA of zebrafish by RT-PCR and constructed 3 types of baff overexpression plasmids： pEGFP-CI-baff, pEGFP-Nl-baff and plRES2-EGFP-baff. Baff protein expression was estimated by in-vitro cell transfection and Western blotting. Results 3 types of baff overexpression plasmids were examined by agarose gel electrophoresis. Western blotting and in-vitro transfection confirmed the expression of Baff-GFP fusion protein. We also obtained the baff over expression zebrafish by microinjection and fluorescence screening. Conclusion All the plasmid we constructed in our research have been estimated and could be used to generate the disease model of baff overexpression zebrafish which can be manipulated for immune-disease research.