牛恒定链与BoLAⅡ类分子β链活性片段的结合与定位分析  

Binding and Localization Analysis Between Bovine(Bos taurus)Invariant Chain and the Active Fragment of BoLA ClassⅡMoleculeβChain

作　　者：陈芳芳[1] 于凤梅 张俊 谈阳 刘翠艳[1] 桂亚萍 CHEN Fang-Fang;YU Feng-Mei;ZHANG Jun;TAN Yang;LIU Cui-Yan;GUI Ya-Ping(Anhui Province Laboratory of Zoonosis,College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China)

机构地区：[1]安徽农业大学动物科技学院/安徽省人畜共患病实验室,合肥230036

出　　处：《农业生物技术学报》2022年第10期1884-1893,共10页Journal of Agricultural Biotechnology

基　　金：安徽省自然科学基金(2108085MC116);安徽省教育厅重点项目(KJ2020A0124)。

摘　　要：牛白细胞抗原(bovine leukocyte antigen,BoLA),又名主要组织相容性复合体(major histocompatibility complex,MHC),其Ⅱ类分子及其伴侣蛋白恒定链(invariant chain,Ii)在加工和递呈抗原肽中起关键作用。为了解在此过程中BoLAⅡβ链与Ii结合和在细胞内定位的特征,本研究提取了淮北黄牛(Bos taurus)血细胞mRNA并反转录为cDNA,应用PCR技术扩增牛Ii与BoLAⅡβ链及结构域的基因片段,并分别构建相应的真核和原核表达质粒。利用原核表达系统进行蛋白的表达和纯化,应用拉下法和Western blot检测Ii结合BoLAⅡβ及其片段的结合关系。应用激光共聚焦显微镜观察Ii与BoLAⅡβ链及其片段在细胞内共定位。结果显示,构建的含牛Ii、BoLAⅡβ、BoLAⅡβ(β1β2)和BoLAⅡβ(TC)基因的重组质粒转化细菌,经诱导能正确表达蛋白并得到纯化。BoLAⅡβ和BoLAⅡβ(β1β2)具有结合Ii的活性功能,而BoLAⅡβ(TC)却无此功能。同时,在真核细胞内Ii能与BoLAⅡβ及其主要结构域BoLAⅡβ(β1β2)和BoLAⅡβ(TC)共定位。综上所述,BoLAⅡβ的β1β2是结合Ii的关键结构域,而BoLAⅡβ的β1β2和TC结构域均具有与Ii在细胞内共定位功能。本研究为进一步研究牛BoLA分子与Ii在免疫应答中的转运及其机理提供了重要实验依据。Bovine leukocyte antigen(BoLA),also named major histocompatibility complex(MHC),its classⅡmolecules and chaperone protein,invariant chain(Ii),plays a key role in the processing and presentation of antigenic peptides.In order to understand the characteristics of binding between BoLAⅡβchain and Ii,and intracellular localization in this process,in this study,mRNA from blood cells of Huaibei yellow cattle(Bos taurus)was extracted and reverse transcribed into cDNA,the genes of BoLAⅡβ,its fragments and Ii were amplified by PCR and then the corresponding eukaryotic and prokaryotic expression plasmids were constructed,respectively.The proteins were expressed and purified by prokaryotic expression system.The binding between Ii and BoLAⅡβchain or its fragments was detected by pull-down method and Western blot.The co-localization of Ii with BoLAⅡβand their fragments in cells was observed by laser confocal microscope.The results showed that the recombinant plasmid containing Ii,BoLAⅡβ,BoLAⅡβ(β1β2)and BoLAⅡβ(TC)genes could be induced and purified.BoLAⅡβand BoLAⅡβ(β1β2)had the active function of binding Ii,but BoLAⅡβ(TC)did not.Meanwhile,Ii could co-localized with BoLAⅡβ,BoLAⅡβ(β1β2)or BoLAⅡβ(TC)in eukaryotic cells.In conclusion,theβ1β2 of BoLAⅡβwas the key domain for its intracellular binding and the both domainsβ1β2 and TC were the functional domains of co-localization with Ii in cells.This study provides an important experimental basis for further study on the transport and mechanism of BoLA molecule and Ii in immune response.

关 键 词：牛 恒定链(Ii) 牛白细胞抗原(BoLA) 结合 共定位 

分 类 号：S852.4[农业科学—基础兽医学]