鸡B-FA分子中结合Ii链功能片段特性的研究  

Research of functional segments of chicken B-FA molecule binding with Ii chain

作　　者：喻丹丹[1] 吴琼[1] 罗兰芳[1] 余为一[1] 陈芳芳[1] 

机构地区：[1]安徽农业大学安徽省人兽共患病重点实验室,合肥230036

出　　处：《中国免疫学杂志》2016年第10期1413-1418,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(No.31572496;31372417)

摘　　要：目的:研究鸡Ii链结合的B-FA分子的功能片段及其特征。方法:将克隆的B-FA基因片段(α1α2、sα1α2和α3TC)分别插入原核或真核表达质粒,然后分别转染或与Ii共转染工程菌E.coli(BL-21)或293T细胞,经过诱导表达、亲和层析纯化和SDS-PAGE鉴定后,分别用Pull-down法观察B-FA片段与Ii结合与在细胞内的共定位特征。结果:首先,构建了3个重组原核表达质粒和4个重组真核表达质粒。原核表达的B-FA片段经亲和层析纯化可获得单一蛋白分子。其次,用Pulldown从共转染工程菌表达的蛋白分子中分别检测到Ii/B-FA-α1α2和Ii/B-FA-sα1α2,而未能检测到Ii与α3TC的结合物,而免疫印迹结果也表明该两片段是结合Ii链形成复合物的功能片段。最后,在真核表达的293T中,B-FA-α1α2具有同B-FA相同的细胞定位特性。结论:鸡B-FA-α1α2片段是结合Ii的功能片段,并保持了B-FA的细胞定位的作用。本研究结果首次提供了B-FA分子与Ii的互相作用的实验依据。Objective： To research the functional segments of B-FA molecule binding invariant chain and their characters. Methods： The DNA segments（ α1α2,sα1α2 and α3TC） of B-FA genes were respectively cloned and inserted into prokaryotic or eukaryotic expression plasmids,then they were singly or co-transfected with Ii gene into the engineering bacteria E. coli（ BL-21）or 293 T cells. After induction of expression,affinity chromatography and SDS-PAGE identification,the binding between B-FA segments and Ii molecule and co-localization in cells were observed with Pull-down and Western blot. Results： First three recombinant prokaryotic expression plasmids and four recombinant eukaryotic expression plasmids were constructed. The single molecules expressed by B-FA segments were observed after an affinity chromatography. Secondly the complexes of Ii / B-FA-α1α2 and Ii / B-FA-sα1α2 were detected by a Pull-down from the co-transfected corresponding prokaryotic expression plasmids,but no complex of Ii and α3TC,also in the western blot it was detected that B-FA-α1α2 or B-FA-sα1α2 as functional segment could bind Ii to form complex. Finally in eukaryotic expression 293 T cells B-FA-sα1α2 kept localization,the same as B-FA. Conclusion： Chicken B-FA-α1α2 is function segment to bind with Ii molecule and keeps the location characters same as B-FA. The results of this research first time provide experimental evidence about B-FA functional region binding segment to Ii molecule.

关 键 词：恒定链 B-FA片段 Pull-down 表达 复合物 

分 类 号：S852.4[农业科学—基础兽医学]