摘要
恒定链（Invariant chain，Ii）分子作为MHC II分子的伴侣分子在内质网中促进了MHC II分子正确折叠、九聚体化及靶向内体的转运，在酸性内体中外源性表位与CLIP序列交换并结合到MHC II分子沟槽，进而抗原被呈递到细胞表面。Ii对MHC II分子的靶向机理已被应用到基于Ii的基因疫苗设计，取得了较好免疫效果，为基因疫苗设计开拓了新思路。除此，Ii还作为多种活性分子的受体参与多种生理过程，因此也成为多种疾病治疗的新靶点。但有关Ii的研究主要集中在人类和小鼠，对于禽类Ii结构与功能研究尚处起步阶段，而禽类生产却面临越来越多的病原挑战，禽类疾病防治也需探索新方法。为此我们以鸳鸯鸭（Moscovy duck,Cairina moschata）为研究对象，对其恒定链及其异构体进行基因克隆、生物信息学分析和结构建模，为深入研究禽类恒定链结构功能、进化趋势与应用等积累理论基础，同时对恒定链在鸳鸯鸭不同组织中的定量表达和定位表达特征进行研究，初步探讨了Ii表达与不同组织器官结构、功能之间的关系。
首先，利用RACE等技术成功获得了1188bp的鸳鸯鸭恒定链（MDIi-1，Genebankno. HQ909102）和1377bp的恒定链异构体（MDIi-2）cDNA序列，其中MDIi-2含有189bp插入序列Tg区，从而证明鸳鸯鸭存在两种恒定链异构体形式。生物信息学分析表明MDIi-1编码222个氨基酸，MDIi-1在核苷酸水平、氨基酸水平及空间结构等方面高度保守，与其他物种Ii比对有相同的功能区，在进化上与其他鸟类Ii高度同源。在不同物种Ii各保守功能区中，CLIP区虽然保持着不低于50%的保守性，但不同物种CLIP区也呈现明显的可变性，这可能是系统进化中环境压力下的适应性选择的结果。MDIi-2编码285个氨基酸，其Tg区高度保守，蛋白质高级结构与其他物种Tg区高度相似。但MDIi-2不是Ii的主要表达形式。
第二，以β-actin基因为内参基因，在十二种组织中分别对MDIi-1和MDIi-2进行实时相对定量PCR。结果表明MDIi-1和MDIi-2两种异构体在同一组织中相伴表达，但MDIi-1是主要表达形式，而且表达水平显著高于MDIi-2。MDIi-1和MDIi-2在被检的十二种器官或组织中分别呈现明显差异表达。脾、胸腺、法氏囊等经典免疫器官呈现MDIi-1的高水平表达，可能与担负机体主要免疫功能有关，但机体成年后胸腺和法氏囊MDIi-1表达水平可能随年龄增长而降低。小肠、肝脏、肾脏等器官也呈现MDIi-1的较高水平表达，其中在小肠粘膜表达水平仅次于脾脏表达，MDIi-1在这些非传统免疫器官可能担负重要局部免疫功能。恒定链在不同组织均有表达暗示其对各种组织都是需要的，只是表达量高低与不同组织先天性结构及所承担的主要功能相关。恒定链不同异构体形式在不同组织细胞中呈现基本一致的差异表达还暗示不同组织细胞存在保守的异构体差异表达调节机制。
第三，原核表达MDIi-1，以表达蛋白免疫小鼠制备抗MDIi-1的抗血清。应用冷冻组织切片技术制备鸳鸯鸭消化管各段、肝脏、脾、胸腺、法氏囊、肺、肾、骨骼肌、心肌等组织切片，以抗MDIi-1血清为一抗、Dylight488标记的兔抗鼠IgG为二抗对MDIi-1在多个器官组织中的分布规律进行了荧光免疫组化定位检测。显示消化管各段粘膜层存在MDIi的高表达巨噬细胞、树突细胞等，MDIi在粘膜上皮和固有层腺体上皮细胞呈弱表达且极性分布；肝细胞不表达MDIi，肝板之间存在个体较大的疑似Kupffer细胞、树突细胞等强表达细胞；胸腺组织中T细胞不表达MDIi，由皮质部向髓质部逐渐增多的网状上皮细胞和交错突细胞等呈现MDIi的强表达；脾组织充满大量强表达MDIi的B细胞；MDIi主要在法氏囊粘膜层表达，其中上皮细胞弱表达且极性分布，固有层淋巴滤泡含有大量高表达MDIi的B细胞，B细胞分布由皮质向髓质逐渐增多、表达增强；肺组织各级支气管上皮细胞不表达MDIi，上皮下结缔组织存少量散在强表达MDIi的疑似巨噬细胞；肾实质的间质中散布MDIi表达细胞，血管球和肾小管上皮不表达MDIi；骨骼肌和心肌组织只在肌纤维间结缔组织存在很少量MDIi表达细胞。
最后，用抗鸡Ii血清和抗鸳鸯鸭Ii血清分别和异种禽类Ii之间存在交叉免疫反应的免疫学方法证明了物种间Ii结构的高度保守性。
Invariant chain (Ii) participates in several key processes of the immune system. Inendoplasmic reticulum, Ii acts as molecular chaperon to facilitate MHC II correctly foldedinto (α:β:Ii)3, and directs (α:β:Ii)3being transported to endocytic compartments via thesorting signal in the cytoplasmic tail of Ii. In endosomal compartments the exogenousepitope exchange class-associated Ii-derived peptide(CLIP) and binds to the groove ofMHC II before being presented to the surface of APCs. Several strategies for Ii-mediatedepitope delivery have been used in the gene vaccine designs which have resulted in moreefficient antigen presentation. In addition to participate in antigen presentation, Ii also actsas active molecular receptors which involve in various physical process. Thus Ii is alsotreated as new target for disease therapies. But research always focuses on the Ii of humanand mouse, research on Ii of poultry is in the beginning stage. Because more and morepathogen challenges are faced in poultry production, new methods for disease control andprevention are needed. So Moscovy duck (Cairina moschata) is selected in this study. Thecloning, characterization and homology analysis of muscovy duck Ii (MDIi) are reportedfor further research on the structure, function, evolution tendency and application of Ii inbirds. The tissue-specific quantitative and positioning expression are also detected todiscuss the relationship between Ii expression and tissue structure＆function in the body.
First, the full-length cDNA sequences of MDIi-1(Genebank no. HQ909102) and MDIi-2are obtained by rapid amplification of cDNA ends PCR (RACE).Thus two isoforms of Iiare proved in muscovy duck. The sequence of MDIi-1has1188bp nucleotides andencodes222amino acids. The sequence of MDIi-2, containing a189bp insert sequencenamed as Tg region, has1377bp nucleotides and encodes285amino acids. Bioinformaticsanalysis shows that MDIi-1has high similarity with Ii of other species not only innucleotide and amino acid level, but also in space structure. Characteristic functionaldomains found in Ii of other species, such as cytoplasmic domain, transmembrane domain,CLIP and trimerization domain, are identified in MDIi. MDIi also show highly homologyin evolution with Ii of other birds. Although all functional domains of Ii show highconservation, a slight diversity in CLIP sequence exists among various species. Thediversity in various CLIP may be adaptability of balance selection under environmentalpressure in evolution. The Tg region in MDIi-2is one of the most conservative sequences.The protein structure of Tg is almost same as that of other species.
Second, tissue-specific quantitative expression of MDIi-1and MDIi-2are respectivelydetected by real-time relativity quantitative PCR in twelve tissues of moscovy duck.β-actin was used as internal control. The results show that MDIi-1and MDIi-2aresimultaneously expressed in the same tissue, but MDIi-1is the main form of expressionbecause its expression level was significantly higher than MDIi-2in various tissues. TheMDIi-1are detected in all tested tissues at different levels. Especially, a higher level is found in spleen, thymus and bursa of fabricius, which are traditional immune tissues, thanother tissues. The high expression of Ii maybe relates to immune function for the wholebody. But Ii expression in thymus and bursa of fabricius maybe decrease along with theage when moscovy duck grows up. Small intestine, liver and kidney as non-traditionalimmune organs also presents high level of MDIi-1. The expression of MDIi-1in smallintestine mucosa is only second to the expression in spleen. MDIi-1in these organs cantake an important local immune function. The tissue-specific expression suggests thatMDIi plays an essential role in all tissues and differential expression maybe relates to theinnate structures and essential functions of these tissues. The almost same models ofdifferential expression of two MDIi isoforms in various tissues also suggest that there areconservative mechanisms regulating expression of two MDIi isoforms in these tissues.
Third, MDIi-1protein is obtained by prokaryotic expression and serum anti-MDIi isprepared by immunizating mice. Tissue-slices are prepared from variant parts of primaeviae, liver, spleen, thymus, bursa of fabricius, lung, kidney, muscle and heart by frozentissue slice technology. Fluorescence immunohistochemical techniques are used to detectMDIi positioning expression in tissues with serum anti-MDIi and Rabbit anti-Rat IgGlabeled by Dylight488. Lots of macrophages and dendritic cells are found to stronglyexpress MDIi in mucosa of primae viae, while MDIi is weakly expressed and distributed inpolarity style in epithelium of mucosa and glands epitheliums of lamina propria.Hepatocytes don’t express MDIi, but MDIi are strongly expressed in larger suspectedKupffers or dendritic cells among hepatic cords in liver. T cells don't express MDIi, whileepithelial reticular cells and interdigitating cells, which are gradually increased from thecortex to the medullary in thymus, strongly express MDIi. Many strongly MDIi expressingB cells are full of the tissue of spleen. The MDIi expressing cells mainly lie in mucosalayer in bursa of fabricius. In bursa of fabricius MDIi is weakly expressed and distributedin polarity style in epithelium of mucosa, however, lots of strongly MDIi expressing Bcells lie in many lymphoid follicles of the lamina propria. These B cells is graduallyincreased and the MDIi expressions are strengthened in them from the cortex to themedullary of lymphoid follicles. MDIi is not expressed in bronchial epithelium cells in thelung, but a small number of suspected macrophages under epithelial layer strongly expressMDIi. The MDIi expressing cells sparsely exist in the gap among renal parenchyma;epitheliums of glomerulus and kidney tubules don't express MDIi. Only few cellsexpressing MDIi exist in connective tissue among muscle fibers in muscle and heart.
Finally, cross immune response are carried out between the serum anti-chicken Ii (oranti-MDIi) and the Ii of heterogeneous poultry, so that the highly conservative property ofIi structure is proved by immunological method.
引文
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