恒定链活性片段转运抗原肽途径影响免疫效果的分子机理

Abstract: Invariant chain (Ii) plays an important role in MHC class II molecule presenting antigen peptides. Recently it was found in our research that some Ii segments as a carrier of antigen peptide showed different character in binding MHC II molecules and promoving immune response, but the pathway to transport peptides is not clear. Therefor we will construct chimeras Ii/Ag containing different active segments of Ii and antigen peptides for following researches: First, to know the molecular michanism of interaction between Ii active segments and MHC class II molecules, the affinity of different chimeras with MHC class II molecules will be detected by Biomolecular Interaction Analysis (BIA), and some mutant chimeras will be constructed and the crucial sites affecting the affinity will be ensured. Secondly, the chimeras and Mhc genes will be co-transfected into cells and the tissue slice from the animals immunized with chimera molecules will be stained to know the localization in the interaction between both molecules and the transport pathway in the cells. Finally, the animal experiments will carry out for analysis and comparison of the relactionship among the affinity of the both molecules, pathway transporting antigen peptides and immune effect. Based on the above researches, the michanism of promoving immune effect via the interaction between Ii active segments with MHC II molecules and potential pathway transporting peptide will be elucidated, which will provide the theoretic and applied basic for contriving new vaccine carrier based on immune molecule segments with the specific regulation function.

恒定链(Ii)在MHCII分子提呈抗原肽中起重要作用。项目组最近在Ii活性片段连接抗原肽的研究中发现，Ii不同片段与MHCII分子的结合和影响免疫效果存在不同特性；但尚不清楚其转运抗原肽的途径。为此，本项目拟构建含鸡Ii不同片段和抗原肽嵌合体(Ii/Ag)进行如下研究：首先，用生物分子互相作用分析仪测定嵌合体与MHCII分子的亲和力，并构建突变体判定影响亲和力的位点，探明Ii活性片段与MHCII分子互相作用的分子机理。其次，用这些嵌合体与Mhc基因共转染真核细胞和用嵌合体蛋白免疫动物进行组织切片染色分析，明确其与MHCII分子作用中的细胞定位和转运途径；最后，进行动物实验，分析并比较分子间亲和力、抗原肽转运途径与免疫效果的关系。通过上述研究，将明确Ii活性片段在与MHCII分子互相作用中转运抗原肽的新途径及影响免疫效果的机理，为创制以免疫分子片段为基础的新型疫苗载体提供理论和应用基础。

恒定链不仅在MHCII分子提呈抗原肽中起重要作用，而且还具有免疫调节活性。项目组在发现恒定链分子部分片段增强免疫应答和细胞定位作用的基础上，在本项目中通过构建含Ii不同活性片段和抗原肽嵌合体(Ii/Ag)。在探明其在细胞内携带抗原肽转运过程中新途径及增强免疫应答的机理方面，获得了以下研究成果：一是明确了Ii/Ag嵌合体中Ii活性片段与MHCII分子结合位点；二是分别用标记的Ii/Ag嵌合体与Mhc基因共转染真核细胞，确定了Ii/Ag嵌合体在细胞的定位、转运特性及其与MHCII分子的关系；三是发现了恒定链与MHCII分子和 I类分子的关系，探明了Ii活性片段与MHCII分子作用的分子机理。四是建立了Pull-Down方法，发现原核表达的鸡和草鱼Ii均能与同源MHCI类和II类分子结合。通过上述研究，为进一步研究Ii与MHC分子的关系奠定了新的基础。