Gene Cloning, Molecular Characterization And Tissue-specific Expression Of Invariant Chain In Muscovy Duck (Cairina Moschata)

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.