多态性BoLAⅠα1α2与恒定链结合及在真核细胞共定位

Polymorphic BolAⅠα1α2 binds to constant chains and co-locates in eukaryotic cells

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BoLAⅠα基因序列,应用分子生物学软件进行比对分析;克隆典型BoLAⅠα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BoLAⅠα片段与Ii在细胞内共定位。结果:首先,所克隆的BoLAⅠα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BoLAⅠα1α2α3、BoLAⅠα1α2和BoLAⅠα3基因的原核重组质粒经诱导表达和蛋白纯化,其中,BoLAⅠα1α2α3、BoLAⅠα1α2具有结合Ii的活性功能。最后,在293T细胞内BoLAⅠα1α2α3和BoLAⅠα1α2与Ii共定位,而单一BoLAⅠα3却不能。结论:BoLAⅠα基因具有高度多态性,BoLAⅠα1α2是与Ii结合和胞内共定位的功能片段。

Objective:To demonstrate the polymorphism ofαchain of bovine major histocompatibility complex(BoLA)classⅠmolecule and domain binding constant chain(Ii).Methods:Total 75 BoLA Iαgenes were obtained from three Huaibei cattle and analyzed by molecular biology software;the genes of typical BoLA Iαdomains and Ii were cloned,and then inserted into prokaryotic expression plasmid.After induced protein expression;the domains of BoLAⅠαchain 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 Iαsegments with Ii was observed by laser confocal microscopy.Results:Firstly,it was found that there were at least 5 kinds of BoLA Iαin the cloned gene sequence,which were highly polymorphic and they were mainly distributed in the antigen peptide binding region(PBR)of BoLAⅠ(α1α2)and cytoplasmic region.Secondly,the prokaryotic recombinant plasmids containing BoLAⅠα1α2α3,BoLAⅠα1α2 or BoLAⅠα3 were constructed,then they were respectively induced to express and purified,in which,the BoLAⅠα1α2α3 and BoLAⅠα1α2 had the activity of binding to Ii.Finally,in 293T cells BoLAⅠα1α2α3 or BoLA Iα1α2 was found that could co-localize with Ii,while a single BoLA Iα3 could not.Conclusion:BoLAⅠαgene is highly polymorphic.BoLAⅠα1α2 is a func-tional fragment that binds to Ii and co-locates intracellular.