多态性BoLA Ⅰα1α2与恒定链结合及在真核细胞共定位
Polymorphic BolA Ⅰα1α2 binds to constant chains and co-locates in eukaryotic cells
陈芳芳 1于凤梅 1刘翠艳 1桂亚萍 1李锦春1
作者信息
- 1. 安徽农业大学动物科技学院,合肥 230036
- 折叠
摘要
目的:探明牛主要组织相容性复合体(BoLA)Ⅰ类分子α链的多态性和结合恒定链(Ii)的结构域.方法:从3头淮北黄牛获得75条BoLA Ⅰα基因序列,应用分子生物学软件进行比对分析;克隆典型BoLA Ⅰα结构域和Ii基因插入原核表达质粒,诱导蛋白表达后进行纯化鉴定,用拉下法和免疫印迹检测BoLA Iα链与Ii结合的结构域.构建相应真核重组质粒,应用激光共聚焦显微镜观察其BoLA Ⅰα片段与Ii在细胞内共定位.结果:首先,所克隆的BoLA Ⅰα基因序列存在至少5种基因型,呈现高度多态性,分布于该分子的抗原肽结合区域(α1α2)和胞浆区.其次,构建的含BoLA Ⅰα1α2α3、BoLA Ⅰα1α2和BoLA Ⅰα3基因的原核重组质粒经诱导表达和蛋白纯化,其中,BoLA Ⅰα1α2α3、BoLA Ⅰα1α2具有结合Ii的活性功能.最后,在293T细胞内BoLA Ⅰα1α2α3和BoLA Ⅰα1α2与Ii共定位,而单一BoLA Ⅰα3却不能.结论:BoLA Ⅰα基因具有高度多态性,BoLA Ⅰα1α2是与Ii结合和胞内共定位的功能片段.
Abstract
Objective:To demonstrate the polymorphism of α chain of bovine major histocompatibility complex(BoLA)classⅠmolecule and domain binding constant chain(Ii).Methods:Total 75 BoLA Iα genes were obtained from three Huaibei cattle and analyzed by molecular biology software;the genes of typical BoLA Iα domains and Ii were cloned,and then inserted into prokaryotic expression plasmid.After induced protein expression;the domains of BoLA Ⅰα chain binding to Ii were detected by pull-down meth-od and Western blot.The recombinant eukaryotic expression plasmids were constructed and the co-localization of BoLA Iα segments with Ii was observed by laser confocal microscopy.Results:Firstly,it was found that there were at least 5 kinds of BoLA Iα in the cloned gene sequence,which were highly polymorphic and they were mainly distributed in the antigen peptide binding region(PBR)of BoLA Ⅰ(α1α2)and cytoplasmic region.Secondly,the prokaryotic recombinant plasmids containing BoLA Ⅰα1α2α3,BoLA Ⅰα1α2 or BoLA Ⅰα 3 were constructed,then they were respectively induced to express and purified,in which,the BoLA Ⅰα1α2α3 and BoLA Ⅰα1α2 had the activity of binding to Ii.Finally,in 293T cells BoLA Ⅰα1α 2α3 or BoLA Ⅰα1α2 was found that could co-localize with Ii,while a single BoLA Ⅰα3 could not.Conclusion:BoLA Ⅰα gene is highly polymorphic.BoLA Ⅰα1α2 is a func-tional fragment that binds to Ii and co-locates intracellular.
关键词
BoLA/高度多态性/Ii/结合/共定位Key words
BoLA/Highly polymorphic/Ii/Combination/Co-localization引用本文复制引用
基金项目
国家自然科学基金(31572496)
安徽省自然科学基金(2108085MC116)
安徽省教育厅重点项目(KJ2020A0124)
出版年
2024
国家科技期刊平台
NSTL
万方数据