鸡Ii结合Rab5a和Rab7b分子的分析

本研究旨在探明鸡恒定链（invariant chain，Ii）与内吞体转运蛋白Rab5a和Rab7b结合的结构域和在细胞内共定位的特征。首先，用PCR和基因突变技术将Ii胞浆区与跨膜区Ii_{（Cyt-Tra）}]、Ii CLIP （class Ⅱ-associated invariant chain peptide）-三聚体区Ii_{（CLIP-TRIM）}]和Ii突变体Ii_{（M81-87aa）}、Ii_{（M91-99aa）}和Ii_{（M81-99aa）}]分别插入pET-32a和pEGFP-C1构建相应的原核和真核重组质粒。其次，将构建的含有绿色荧光蛋白的重组质粒与实验室保存的含有红色荧光Rab5a和Rab7b的重组质粒共转染至人胚胎肾细胞系293 T，观察它们的共定位。将构建的原核重组质粒进行表达和纯化，最后用拉下法和免疫印迹检测Ii与Rab5a和Rab7b的结合域。结果表明，成功构建Ii结构域及Ii突变体的重组质粒。Ii_{（Cyt-Tra）}及Ii突变体均能与Rab5a和Rab7b在细胞内共定位，而Ii_{（CLIP-TRIM）}与空载体却不能。Ii的胞浆区和跨膜区是与Rab5a和Rab7b结合的功能结构域，而不是CLIP与三聚体区。综上所述，鸡Ii与Rab5a和Rab7b共定位和结合的区域是其胞浆区和跨膜区，而不是内质网腔区。这些结果提示Rab分子参与了Ii在胞内细胞器的转运机制，为进一步研究Ii及其载体在细胞内的转运机制和功能提供了新的途径。

Analysis on Ii Binding Rab5a and Rab7b Molecules

This study aimed to investigate the binding domain and intracellular localization of chicken invariant chain (Ii) to the endosome transporters Rab5a and Rab7b. First, the cytoplasmic region and transmembrane region (Ii_(Cyt-Tra)), Ii CLIP (class Ⅱ-associated invariant chain peptide) with trimer region (Ii_(CLIP-TRIM)) and Ii mutants (Ii_(M81-87aa),Ii_(M91-99aa)and Ii_(M81-99aa)) obtained by PCR and gene mutation techniques were inserted into pET-32a and pEGFP-C1 to consturct prokaryotic and eukaryotic recombinant plasmids, respectively, the corresponding eukaryotic and prokaryotic recombinant expression plasmids were constructed. Secondly, the constructed recombinant plasmid containing green fluorescent protein and the recombinant plasmids containing red fluorescent Rab5a and Rab7b were co-transfected into human embryonic kidney cell line 293 T, and their co localization was observed. Finally, the binding domain of Ii with Rab5a and Rab7b was detected by pull-down method and Western blot. The results showed that the recombinant plasmids of Ii domain and mutant were successfully constructed. Both Ii _(cyt-tra) and Ii mutants could co localize with Rab5a and Rab7b, but Ii _(CLIP-TRIM) and empty vector could not. The cytoplasmic and transmembrane domains of Ii are the main domains binding Rab5a and Rab7b, not CLIP and trimer domains. In conclusion, the colocalization and binding regions of chicken Ii with Rab5a and Rab7b are cytoplasmic and transmembrane regions, not endoplasmic reticulum cavity regions. These results further suggest that Rab molecule is involved in the transport mechanism of Ii in intracellular organelles, and provide a new way for further study of the transport mechanism and function of Ii and its carrier in cells.