鸡Ii与B-L基因表达产物的细胞定位与结合活性特征  

Characters of location in cells and binding activity of chicken Ii and B-L gene expressed products

作　　者：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》2015年第7期879-883,889,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(No.31201888;31372417)

摘　　要：目的:研究鸡Ii与B-L基因表达产物在细胞内定位和互相结合的活性特征。方法:将克隆的Ii、B-LA和B-LB基因片段分别插入原核或真核表达质粒;然后分别单独转染或共转染工程菌Rosetta(DE3)或293T细胞。所有重组菌经鉴定后进行诱导表达后再复性。单一表达产物用免疫印迹检测它们的抗原性,共转染表达的产物用pull-down法和(SDS-)PAGE观察其互相结合特征。结果:成功构建了6个原核和真核表达重组质粒。在真核细胞中Ii、B-LA和B-LB基因表达产物定位于细胞浆膜,而原核表达产物经复性后和亲和层析纯化,获得单一蛋白分子。其次,原核表达的Ii与B-LA和B-LB复性蛋白能够诱导产生鼠源抗体,这些抗体特异性结合真核表达产物,表明它们保持相同的免疫原性。最后,用pull-down从共转染的工程菌表达并复性的蛋白分子中分别获得纯化的Ii/B-LA和Ii/B-LB复合物,它们经SDS处理后分别被解离成单体。结论:鸡Ii和B-L基因的原核与真核表达的产物能够保持其抗原性,而原核共表达的Ii和B-L蛋白分子复性后可以互相结合。本研究的结果为进一步研究Ii与B分子关系提供了方法。Objective：To study characters of the location in cells and binding activity of chicken Ii and B-L gene expressed products. Methods： The cloned gene segments of chicken Ii,B-LA and B-LB were respectively inserted into prokaryotic or eukaryotic expression plasmids,and then these recombinant plasmids were respectively alone transfected or cotransfected into engineering bacteria,Rosetta（ DE3） or 293 T cells. All of the recombinant bacteria were induced to express and their products then were renatured. The singly expressed products were detected to their immunogenicity with Western blot,and the co-expressed products were tested their binding with pull-down method and（ SDS-） PAGE. Results： First six of prokaryotic and eukaryotic recombinant expression plasmids were constructed. The eukaryotic expressed products of Ii,B-LA and B-LB genes located in cellular plasma membrane. The single protein molecules were achieved from prokaryotic expressed products,which were renatured and purified with a Ni-column respectively. Secondly the prokaryotic expression Ii,B-LA and B-LB molecules could respectively induce mouse to product specific antibodies,which could recognize the corresponding products in eukaryotic expression. This indicated that they retained their antigenicity. Finally with pull-down from the products in co-expression in engineering bacteria and renaturation the Ii / B-LA or Ii / B-LB complexes were purified and the Ii,B-LA or B-LB monomers were dissociated from these complexes after a SDS treatment. Conclusion： The prokaryotic or eukaryotic expressed products of chicken Ii and B-L genes could retain their antigenicity,and chicken Ii could bind B-L molecules after prokaryotic coexpression and renaturation. These results provide a useful method to study the relation between Ii and MHC molecules.

关 键 词：恒定链 B-L Pull-down 表达 复合物 

分 类 号：S852.4[农业科学—基础兽医学]