鸡Rab5a与恒定链的结合特性及其在真核细胞的共定位研究  

Study on the Binding Characteristics of Rab5a with Invariant Chain of Chicken(Gallus gallus)and Their Co-localization in Eukaryotic Cells

作　　者：谭红黎 桂亚萍 李梅珍 黄鑫 秦文娟 查丽莎[1] 陈芳芳[1] TAN Hong-Li;GUI Ya-Ping;LI Mei-Zhen;HUANG Xin;QIN WEN-Juan;ZHA Li-Sha;CHEN Fang-Fang(Animal Molecule and Applied Immune Innovation Team of Anhui Agricultural University,Hefei 230036,China)

机构地区：[1]安徽农业大学动物分子与应用免疫创新团队,合肥230036

出　　处：《农业生物技术学报》2020年第6期1021-1029,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31572496);国家重点研发计划专项经费(2018YFD0502003)。

摘　　要：Rab5a是胞内转运蛋白,而恒定链(invariant chain,Ii)则是协助主要组织相容性复合体(major histocompatibility complex,MHC)Ⅱ分子装配成熟以及递呈抗原肽的免疫分子。长期以来,Ii的胞内途径机制并不清楚。本研究旨在探明鸡(Gallus gallus)Rab5a的结构特征及参与Ii在胞内转运中的作用。首先,用自行设计的引物克隆鸡Rab5a基因(GenBank No.XM015281490),构建含鸡cIi、cIi突变体(IiD81-87aa和IiD91-99aa)的重组表达载体;其次,应用软件比对鸡和小鼠(Mus musculus)Rab5a蛋白质结构。最后,分别将Rab5a和cIi转染小鼠树突状细胞系DC2.4细胞,应用免疫荧光技术和激光共聚焦显微镜观察鸡Rab5a与cIi和c IiD81-87aa和cIiD91-99aa在真核细胞内的定位与共定位。应用GST pull-down和Western blot检测鸡Rab5a和Ii蛋白分子间的相互作用。结果表明,克隆获得的鸡Rab5a基因与预期大小一致,为648 bp。分子结构分析表明,鸡与小鼠的Rab5a高度相似,同源性高达95.4%。同源建模分析结果显示,鸡与小鼠Rab5a均含有5个α螺旋和6个β折叠区,表明哺乳类和禽类Rab5a是同源的。激光共聚焦显微镜观察表明,鸡Rab5a和Ii均定位于细胞的早期内吞体,而突变体IiD81-87aa和IiD91-99aa却不能定位于早期内吞体。GST pull-down和Western blot结果显示鸡Rab5a能够结合Ii分子。综上所述,鸡Rab5a与Ii在早期内吞体的共定位和互相结合提示,Rab5a参与Ii在细胞内的转运。本结果为进一步研究其机理提供了新依据。Rab5a molecule is an intracellular transporter,while the invariant chain(Ii)is an immune molecule that helps MHC(major histocompatibility complex)to assemble,mature and present antigen peptide.For a long time,the intracellular pathway mechanism of Ii is not clear.The purpose of this study was to explore the structural characteristics of Rab5a in chicken(Gallus gallus)and its role in the intracellular transport of Ii.First,chicken Rab5a gene was cloned with self-designed primer,and the eukaryotic expression recombinant vectors containing chicken cIi and its mutants(IiD81-87aa and IiD91-99aa)were constructed.Secondly,the protein structure of Rab5a in chicken and mouse(Mus musculus)were compared by software.Finally,Rab5a and Ii were transfected into mouse dendritic cell line DC2.4 cells,respectively.The localization and co-localization of chicken Rab5a and Ii,cIiD81-87aa and cIiD91-99aa in eukaryotic cells were observed by immunofluorescence and laser confocal microscope.The results showed that the Rab5a gene was 648 bp,which was consistent with the expected size.The molecular structural analysis showed that Rab5a of chicken was highly similar to that of mouse,and the homology was as high as 95.4%.The results of homologous modeling analysis showed that Rab5a of chicken and mouse contained 5 alpha helixes and 6 beta helixes,indicating that Rab5a of mammal and poultry were homologous.Laser confocal microscope observation showed that chicken Rab5a and Ii could be localized in the early endocytosis of cells,but the mutants IiD81-87aa and IiD91-99aa could not be localized in the early endocytosis.The results of GST pull-down and Western blot showed that Rab5a could bind Ii molecule.In conclusion,the results of co-localization and binding of Rab5a and Ii in the early endocytosis suggest that Rab5a is involved in the transport of Ii in cells,which provides a new basis for further study of its mechanism.

关 键 词：RAB5A Ii转运 细胞内吞体 细胞器定位 

分 类 号：S852.4[农业科学—基础兽医学]