鸡恒定链协助抗原肽结合MHCⅡ类分子并进入细胞内吞体

Chicken Ii Helps Antigen Peptide Bind MHC ClassⅡ Molecules and Enter Endosomes

为探明鸡恒定链(invariant chain,Ii)的胞质区/跨膜区[Ii(Cyt/Tra)]作为载体是否可携带抗原肽在细胞内结合MHCⅡ类分子和进入转运途径的细胞器(内吞体),构建4个基因目的片段[Ii、Ii(Cyt/Tra)、F2(新城疫病毒F蛋白片段)和Ii(Cyt/Tra)/F2],分别将它们插入原核表达载体pET-32a和真核表达载体pmCherry-C1/N1中,构建8个重组质粒,再转入工程菌(E.coli)和人肾细胞系(293T),并应用拉下法(pull-down)检测目的蛋白与MHCⅡβ的结合,应用激光共聚焦确定它们在真核细胞与MHCⅡβ的共定位以及在内吞体的定位。结果表明,构建的重组质粒均能相应地原核或真核表达相应目的蛋白;Ii、Ii(Cyt/Tra)和Ii(Cyt/Tra)/F2不仅能够与MHCⅡβ结合,还能进入细胞内吞体;但是F2既不能与MHCⅡβ结合,也不能进入内吞体。Ii活性片段Cyt/Tra不仅本身具有结合MHCⅡβ的作用,而且还可以携带抗原肽与之结合并一起转入细胞内吞体而进入抗原递呈途径。该结果为进一步研究Ii载体转运抗原提供了理论依据。

This experiment was conducted to clear if chicken invariant chain(Ii)cytoplasmic/transmembrane domains-Ii(Cyt/Tra),as immune carrier-is able to carry antigen peptide to associate with MHC classⅡ molecules and enter the endosomes in cell transport pathway.In this study,4 gene segments,such as Ii,Ii(Cyt/Tra),F2(Newcastle disease virus F protein fragment)and Ii(Cyt/Tra)/F2,were amplified and constructed by PCR.They were inserted into prokaryotic expression plasmid pET-32a and eukaryotic expression plasmid pmCherry-C1/N1 respectively,and 8 recombinant plasmids were constructed.Then they were transferred into the engineering bacteria(E.coli)and human renal cell(293 T)respectively.The binding of the target protein to the MHCⅡβ was detected by pull-down assay,and the co-localization in the eukaryotic cells with the MHCⅡβ and the localization in the endosomes were determined by laser confocal.The results showed that all the recombinant target protein molecules were expressed in prokaryotic or eukaryotes;Ii,Ii(Cyt/Tra)and Ii(Cyt/Tra)/F2 were able to bind to MHCⅡβ as well as to enter endosomes except F2.To sum up,the active domains,Ii(Cyt/Tra),not only itself had the effect of associating MHCⅡβ,but also could bring antigenic peptides to bind MHCⅡβ and transport the endosome together,and finally enter the antigen presentation pathway.This provides a theoretical basis for further study of Ii as immune carrier.