Genetic And Biological Function Analysis Of Fragments Of The Pheasant Immunoglobulin Y Heavy Chain Constant Region

In mammals, immunoglobulins(Igs) are divided into five major classes, Ig M, Ig G, Ig A, Ig D, and Ig E, which are classified based on antigenic differences in the heavy-chain constant regions. Only three classes of antibodies, Ig M, Ig A, and Ig Y have been found in birds. The study and diagnosis of infectious diseases in ring-necked pheasant(Phasianus colchicus) is difficult because of the scarcity of commercially available species-specific anti-pheasant immunoglobulin Y(Ig Y) antibodies. Ig Y is the major serum antibody of amphibians, reptiles, and birds, and it might be the evolutionary ancestor of mammalian Ig G and Ig E. Ig Y consists of two heavy(H) and two light(L) chains. The H chain includes four constant(C) domains, and its length is similar to that of the mammalian Ig E H chain. In contrast to mammalian Ig G, avian Ig Y contains one additional domain in the constant region of the H chains(designated as upsilon, υ) and lacks a hinge region.Many endogenous peptides are known to exert specific regulatory effects on certain functions of the immune, endocrine, nervous, and other systems of humans and other vertebrate species. Such regulatory peptide sequences have been found in human Ig G, including tuftsin, rigin, peptide p24, immunorphin, and immunocortin, and they are thought to act as signaling factors that either stimulate or inhibit the immune response. However, information on the functions of immune peptides of avian Ig Y is scarce. The study focused on the genetic and biological function analysis of fragments of the pheasant immunoglobulin Y heavy chain constant region. This research consists of four parts as follows: Part Ⅰ, Cloning, expression and sequenc analysis of pheasant Ig Y CH fragments.Primers were designed based on chicken and duck Ig Y sequences(Gen Bank accession numbers X07174 and AJ517505, respectively), total RNA was extracted from spleen samples of 20-week-old female pheasants and reverse transcribed to c DNA using the Prim Script RT reagent kit. Three c DNA fragments of approximately 750 base pairs(bp), 478 bp, and 318 bp were obtained. The 750-bp c DNA fragment contained the joining segment(JH) and the CH1, CH2, and partial CH3 regions; the 478-bp c DNA fragment contained the CH2 and CH3 regions; and the 318-bp segment contained the CH1 region. The three fragments encoded proteins of 250, 159, and 106 amino acids, respectively. The Ig Y heavy chain constant region from pheasant showed the highest homology with that from chicken(71.2%). Phylogenetic analysis for Ig Y showed that pheasant was closely related to chicken and duck than to any other analyzed vertebrate species. Part Ⅱ, immunological cross-reactivity assay。The cross-reactivity of Ig G or Ig Y sequences from different vertebrate species was identified by dot-enzyme-linked immunosorbent assay. Strong immune cross-reaction was noted between chicken and pheasant; dog and raccoon dog, fox, and mink. Weak or no crossreactivity was observed between birds and mammals. Western blot analysis showed that the expressed recombinant proteins clearly reacted with rabbit anti-chicken Ig G. These results revealed that rabbit anti-chicken Ig G cross-reacted with the recombinant proteins r Ph Cυ-750 and r Ph Cυ-478. Rabbit anti-chicken Ig G did not have cross-reactivity with the recombinant protein r Ph Cυ-318. Part Ⅲ, biological function assay of CH-peptides of the pheasant immunoglobulin Y heavy chain constant region.The CH-peptides derived from the deduced amino acid sequence of pheasant Ig Y CH cloned in this study using ABCpred server were synthesized by Sangon Biotechnology Co. and used for investigating the immunoregulatory activities of Ig Y in vitro. Chicken peripheral blood monocytes suspensions(1 × 106 ml-1 cells) were treated with CH-peptides at concentrations of 5, 10, 15, and 20 μg ml-1 and incubated at 37 °C in 5% CO2 for 18 h. Our results revealed that T10 L, V10 D, R12 V and A10 Y increased the production of IL-1β, IL-4, and IFN-γ by monocytes, CH-peptides play a potential role as immunoregulants in avian species. Part Ⅳ, the subcellular localization of pheasant Ig Y-Fc peptide and its binding to cellsBy confocal laser scanning microscopy to research the subcellular localization of FITClabeled pheasant Ig Y-Fc peptides in chicken peripheral blood monocytes, and pheasant Fc peptide binding to chicken peripheral blood mononuclear cells was quantified by flow cytometry testing. Chicken peripheral blood mononuclear cells were treated with FITClabeled pheasant Ig Y- Fc peptide for 1h, Fc peptide mainly concentrated on the cell membrane, nucleus and cytoplasm substantially have no fluorescence excitation. The combined rate of A10L(AKLKCLVVNL), R10Q(RFIQRTLQKQ) and A10Y(AIPPSPGELY) with the cells was respectively 96.8%, 97.5% and 96.3%, R10 Q combined with cell was the highest.Chicken peripheral blood mononuclear cells were treated with FITC-labeled pheasant Ig Y- Fc peptide for 18 h, Fc peptide mainly enriched in the nucleus region, cytoplasm and cell membrane substantially have no fluorescence excitation.