鸡MHC Ⅰ分子单链三聚体真核表达载体的构建和表达
Construction and Expression of Single Chain Trimer of Chicken MHC Ⅰ Molecular
戴银 1单文杰 2胡晓苗 1赵瑞宏 1侯宏艳 1沈学怀 1潘孝成 1周学利 1张丹俊1
作者信息
- 1. 安徽省农业科学院畜牧兽医研究所,安徽合肥230031
- 2. 安徽农业大学动物科技学院,安徽合肥230036
- 折叠
摘要
[目的]探索主要组织相容性复合体Ⅰ类分子(Major Histocompatibility Complex,MHC Ⅰ)提高抗原呈递效率的方法,获得具有免疫学活性的抗原肽-MHC Ⅰ分子单链三聚体.[方法]采用基因重组技术,通过连接肽(G4S)3将鸡新城疫病毒抗原表位短肽HN(aa346-353)与鸡MHC Ⅰ(B-F)分子二聚体的编码基因共价串联,构建带有绿色荧光标签蛋白的真核表达载体pEGFP-HN346-353-B-Fβ2m-α.[结果]转染真核细胞293T后,荧光显微镜观察可见,重组融合蛋白HN346-353-B-Fβ2m-α分子三聚体主要表达定位于真核细胞的内膜系统.Western Blot结果表明,在预期蛋白分子量大小位置有明显的特异反应带,重组蛋白与其相应抗体可发生特异性结合反应.[结论]重组质粒能够在真核细胞中表达,且融合蛋白具有良好的生物学活性.
Abstract
[Objective] To improve the efficiency of antigen presentation of MHC Ⅰ molecule,the single chain trimer of pepide-MHC Ⅰ molecules were obtained.[Method] The chicken MHC Ⅰ (B-F) gene and the epitope protein HN (aa346-353) of Newcastle disease virus were linked by a linker with sequences encoding(G4S) 3.Then the recombinant plasmids (pEGFP-HN346-353-B-F β2m-α) with green fluorescent protein was constructed.Further the recombinant plasmid was transfected into eukaryotic cell 293T.[Result] The gene product of recombinant plasmid was mainly located to endomembrane system of the cells by fluorescence microscopy.Moreover,the positive reactions were observed by the method of Western Blot,in accordance with the target proteins,and the proteins had the binding reaction with specific antibodies.[Conclusion] The recombinant plasmid was expressed in eukaryotic cells with a good biological activity.
关键词
鸡MHC/B-F分子/单链三聚体/真核表达Key words
Chicken MHC B-F molecule/Single chain trimer/Eukaryotic expression引用本文复制引用
基金项目
国家自然科学基金(31302044)
国家蛋鸡产业技术体系项目(CARS-41)
安徽省科技攻关项目(11010302119)
出版年
2017
国家科技期刊平台
NSTL
万方数据