【作者】 叶红；

【导师】 余为一；

【作者基本信息】 安徽农业大学 ， 微生物学， 2009， 博士

【摘要】 在免疫系统中,主要组织相容性复合物（Major histocompatibility complex,MHC）Ⅰ、Ⅱ分子作为肽受体,具有提呈抗原肽的功能。恒定链（Invariant Chain,Ii）是一种非多态性Ⅱ型跨膜糖蛋白,它是MHCⅡ分子生物合成及MHCⅡ分子结合外源性抗原肽过程中重要的分子伴侣。研究表明,在内质网中Ii辅助MHCⅡ亚单位初始折叠和低聚化,并与MHCⅡ分子组装成（αβIi）₃九聚体,同时Ii的CLIP区（the class-Ⅱassociated Iichain peptide）即第81-104位氨基酸片段,占据MHCⅡ的抗原结合凹槽,有效屏蔽了MHCⅡ类分子在内质网中与内源性抗原肽结合。细胞中缺乏Ii会导致MHCⅡα链和β链不能正确折叠,从而被较长时间滞留于内质网中。迄今为止,Ii与MHCⅡ分子相互关系的研究多集中在哺乳动物,有关禽类的研究还未见报导。从推测的氨基酸序列分析看,人、小鼠、大鼠的CLIP区（尤其是91-99aa:MRMATPLLM）是完全保守的,但这种高度的保守性未出现包括禽类在内的其他物种中。对于鸡Ii与鸡Ⅱ类抗原递呈分子亚单位在细胞中的定位如何,Ii链能否与MHCⅡα和MHCⅡβ的单链聚合,Ii链中的CLIP区在聚合中起何作用,外源性抗原肽替代CLIP后,Ii链是否还能与MHCⅡ链聚合?为了探讨这些问题,我们构建了表达红色荧光蛋白（Red Fluorescent Protein,RFP）的鸡Ⅱ类抗原递呈分子亚单位即MHCⅡα链和β链的真核表达载体和表达增强型绿色荧光蛋白（Enhanced Green Fluores cenceProtein,EGFP）的Ii真核表达载体,使用瞬时表达系统,检测Ii与单个MHCⅡ亚单位在COS-7细胞中的表达和定位,并通过免疫共沉淀进一步研究它们之间的相互关系。研究表明,带有荧光蛋白的融合蛋白保持了目的蛋白（Ii、MHCⅡα和MHCⅡβ）的活性和标记蛋白（GFP和RFP）的特性且定位于细胞的内膜系统;其次鸡Ii链与单链MHCⅡα或MHCⅡβ之间存在细胞内的共定位,并且它们之间能够形成复合体,在这一过程中CLIP区担当关键角色。为了探索研制高效的核酸疫苗,我们将以新城疫病毒的F₃₄₃抗原表位基因（aa327-359）替换鸡Ii链CLIP编码基因构建的Ii嵌合体分别与单个MHCⅡ亚单位共转染COS-7细胞,通过荧光显微镜观察Ii嵌合体改变了抗原表位在细胞中的定位,并且通过免疫共沉淀证实用NDV的F₃₄₃抗原肽取代CLIP区的Ii嵌合体保持与单链MHCⅡα或MHCⅡβ聚合。这些结果提示,只要保持Ii分子基本的空间结构,Ii链就能与MHCⅡα或MHCⅡβ链形成聚合体。因此,在新型高效基因疫苗的设计中,Ii可以充当抗原肽潜在的运输载体将抗原肽靶向性地传递到MHCⅡ递呈抗原分子的路径中。病毒性抗原作为内源性抗原由MHCⅠ类分子呈递,而Ii分子作为MHCⅡ分子的伴侣蛋白主要协助递呈外源性抗原。MHCⅠ类分子和MHCⅡ类分子的合成均起源于内质网,它们在细胞中的分选则发生在高尔基体的反面膜囊,MHCⅠ类分子通过膜泡运输离开高尔基体到达细胞表面,MHCⅡ类分子则进一步到达溶酶体。MHCⅠ与MHCⅡ所走路线的不同可能与Ii的引导有关。为了了解鸡的Ii分子与MHCⅠ类分子之间的关系,本文还探讨了它们之间是否存在相互作用。为此,我们首先获得了鸡MHCⅠ分子亚单位的基因,并进一步构建了相应的原核表达质粒,用pET-32a和Rosseta菌组合表达带有信号肽的MHCⅠα、MHCⅠβ_2m。其次,我们构建了能表达红色荧光蛋白的鸡MHCⅠ类抗原递呈分子亚单位的真核表达载体,并与能表达增强型绿色荧光蛋白的Ii真核表达载体,使用瞬时表达系统共转染COS-7,荧光显微镜检测与免疫共沉淀的结果表明,单个的鸡MHCⅠα重链或β_2m轻链均不能与鸡Ii有效结合,Ii分子要求与完整的MHCⅠ分子结合才能形成复合体。更多还原

【Abstract】 Major histocompatibility complex（MHC） classⅡmolecules are polymorphic cell surface glycoproteins that serve the immune system as peptide receptors.The invariant chain（Ii） is a non-polymorphic typeⅡtransmembrane protein and acts as a chaperone during MHC classⅡ（MHCⅡ） biosynthesis and binding foreign peptide antigens.In biosynthesis Ii assists initial folding and oligomerisation of the MHCⅡsubunits.The final assembled classⅡ-Ii complex is a nonamer,consisting of a single Ii trimer associated with three classⅡheterodimers.The association of Ii with MHC classⅡmolecules involves the direct occupancy of the peptide-binding groove with a region of Ii known as the CLIP （classⅡ-associated invariant chain peptide,amino acids 81-104） domain.Occupancy of the classⅡpeptide binding site with CLIP plays an important role in preventing MHC classⅡmolecules from the loading of endogenetic antigenic peptides in the endoplasmic reticulum（ER）.In the absence of Ii,unassembled MHCⅡαandβchains are largely retained within ER.So far,the studies on the association of MHCⅡwith Ii have been mainly focusing on mammal,especially human,and have never been shown in poultry.Analysis of the putative amino acid sequence of Ii indicated that the CLIP region（especially aa 91-99: MRMATPLLM） is completely conserved between human,mouse and rat.But this striking conservation does not extend to other species,including poultry.Whether can chicken Ii associate with single MHCⅡαorβchain and what does its CLIP play? Can the Ii replaced CLIP with exogenous antigens peptides associate with MHCⅡsubunits? For studying these questions,we respectively constructed the eukaryotic expression plasmids pEGFP-C1-Ii,pDsRed2-N1-α,pDsRed2-N1-βthat fused red or enhanced green fluorescent protein.The recombinant plasmids were transiently transfected into COS-7 cells with Lipofectamine 2000.Immunofluorescence microscopy was carried out to detect the expression and cellular localization of Ii and single MHCⅡsubunit,and immunoprecipitation was used for analyzing the association of chicken Ii orΔCLIP- Ii with the MHCⅡsubunits.The results indicated that the fusion genes ofα,βor Ii was predominantly expressed and localized in cellular endomembrane system,and changed the cytoplasm localization of the fluorescent protein.Chicken Ii with individual MHCⅡαorβpolypeptides colocalized in cellular endomembrane system,and its CLIP region plays a key role on the assembly of Ii with MHCⅡsubunits.Furthermore,we also found that the Ii replaced CLIP with Newcastle disease virus F₃₄₃ epitopes(NDV F₃₄₃) can not only efficiently load F₃₄₃ antigenic peptides into the vesicles of endomembrane system but also. restore the association with MHCⅡsubunits in the transfected cells.Thus,MHCⅡpolymer assembly is not blocked as long as the basic steric molecular structure of Ii is maintained,and different binding models exist in different species or MHCⅡisotypes. Moreover,in the designing of effective DNA vaccines,chicken Ii could act as a potential "carrier" of the antigenic peptides for the delivery of an antigenic peptide to the MHC classⅡpathway.Both MHCⅠand MHCⅡmolecules are synthesized in the ER,yet they were sorted in trans-Golgi.MHCⅠmolecules were transported to the cellular surface by vesicles,and MHCⅡmolecules must be transported to lysosome.The different sorting route of MHCⅠand MHCⅡmolecules may be related to the guidance of Ii.To investigate the intracellular localization and association of chicken major histocompatibility complex classⅠsubunits with invariant chain,we carried out a series of the experiments.At first,the sequences of MHCⅠαandβ_2m subunits were amplified with RT-PCR,and sequentially the prokaryotic expressive vector pET-32a-MHCⅠαand pET-32a-MHCⅠβ_2m containing signal peptides was constructed and expressed by Rosetta bacteria.Secondly,we constructed the eukaryotic expression plasmids of MHCⅠαand MHCⅠβ_2m that fused red or enhanced green fluorescent protein,respectively.The recombinant plasmids of MHCⅠsubunits and pEGFP-C1-Ii that can express enhanced green fluorescent protein were transiently transfected into COS-7 cells.The colocalization was found in endocytic compartments when GFP-Ii,MHCⅠα-RFP and MHCⅠβ_2m-RFP were co-expressed in COS-7 cells. Immunoprecipitation of Ii showed that GFP-Ii co-isolated with MHCⅠα-GFP and MHCⅠβ_2m-GFP subunits when COS-7 cells were transiently transfected with pEGFP-C1-Ii, pEGFP-N1-MHCⅠαand pEGFP-N1-MHCⅠβ_2m.Thus,Chicken Ii can not effectively associate with singleαorβ_2m subunits from chicken MHCⅠmolecules,but Ii and the integrated MHCⅠmolecule can form the complexes that are colocalized in endomembrane system of COS-7 cells.更多还原