Abstract：This work focus on clearing active segments of chicken (Gallus gallus) major histocompatibility complex (MHC) class Ⅱmolecules binding with invariant chain (Ii) for research of mechanism of MHC and Ii action in presenting antigen peptides. To this end 3 DNA segments (Sβ1, β1 and β2TC) were cloned from cDNA of B-LB gene, which was kept in our laboratory, and inserted into prokaryotic or eukaryotic expression plasmids respectively. Then these recombinant plasmids were respectively transfected or co-transfected with Ii into engineering bacteria, Rosetta (DE3). The expression products or complexes (B-LB segment and Ii) were purified by affinity chromatography and identified by pull-down and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, the expression and localization of B-LB segments and Ii in the eukaryotic cells were observed too. The results showed that first the 3 recombinant plasmids (pGEX-4T-1-B-LB-Sβ1, pGEX-4T-1-B-LB-β1 and pGEX-4T-1-B-LB-β2TC) could well express, and the interest proteins (B-LB-Sβ1, B-LB-β1 and B-LB-β2TC) also well be purified by an affinity chromatography. Secondly the segment B-LB-Sβ1 or B-LB-β1 rather than B-LB-β2TC bound His/Ii, which adsorbed to Ni-column, forming complexes by the co-transfection and followed pull-down, because in the Western blot the 3 interest proteins could be recognized by specific antibody in the co-transfected products, but only B-LB-Sβ1 and B-LB-β1 were found as the complexes (His/Ii and B-LB-Sβ1, His/Ii and B-LB-β1), which were dissociated from Ni-column in pull-down. Finally it was proved that B-LB-Sβ1 and B-LB-β1 could express and localize in the endometrial system of 293T cells, which kept the function such as whole MHC molecule, but the B-LB-β2TC lost this function. All of above results first time provide an evidence of binding of active segments of MHC class Ⅱmolecules with Ii in vitro.
罗兰芳,余为一,陈芳芳. 2015. 鸡Ii与B-L基因表达产物的细胞定位与结合活性特征[J].中国免疫学杂志,31(7):879-883.(LUO L F, YU W Y, CHEN F F. 2015.Characters of the location in cells and binding activity of chicken Ii and B-L gene expressed products[J].Chinese Journal of Immunology, 31(7):879-833.)
Biedrzycka A, Radwan J. 2008. Population fragmentation and major histocompatibility complex variation in the spotted suslik, Spermophilus suslicus[J]. Molecular Ecology, 17(22):4801-4811.
Chaves L D, Krueth S B, Reed K M. 2009. Defining the turkey MHC: sequence and genes of the B locus[J]. The Journal of Immunology, 183:6530-7537.
Chen F F, Pan L, Wu C, et al.2012. Character of chicken polymorphic MHC class II alleles of three Chinese local breeds[J]. Poultry Science, 91:1097-1104.
Chen F F, Wu C, Pan L, et al.2013. Cross-species association of quail invariant chain with chicken and mouse MHC II molecules[J]. Developmental Comparative Immunology, 40(1):20-27.
Dixon A M, Stanley B J, Matthews E E, et al.2006. Invariant chain transmembrane domain trimerization: a step in MHC class II assembly [J]. Biochemistry, 45:5228-5234.
Eizaguirre C, Yeates S E, Lenz T L, et al.2009. MHC-based mate choice combines good genes and maintenance of MHC polymorphism[J]. Molecular Ecology,18(15):3316-3329.
Frauwirth K, Shastri N. 2001. Mutation of the invariant chain transmembrane region inhibits II degradation, prolongs association with MHC class II, and selectively disrupts antigen presentation [J]. Cellular Immunology, 209:97-108.
Gelin C, Sloma I, Charron D et al.2009.Regulation of MHC II and CD1 antigen presentation: from ubiquity to security[J]. Journalof Leukocyte Biology, 85(2):215-224.
Gunther S, Schlundt A, Sticht J, et al. 2010. Bidirectional binding of invariant chain peptides to an MHC class II molecule[J]. ProceedingsoftheNational Academy of SciencesUSA,107:22219-22224.
Gu X, Nei M. 1999. Locus specificity of polymorphic alleles and evolution by a birth-and-death process in mammalian MHC genes[J]. Molecularand Biological Evology, 16(2):147-156.
Hughes C R, Miles S, Walbroehl J M. 2008. Support for the minimal essential MHC hypothesis: a parrot with a single, highly polymorphic MHC class II B gene[J]. Immunogenetics, 60(5):219-231.
Hughes A L, Friedman R. 2008. Genome size reduction in the chicken has involved massive loss of ancestral protein-coding genes[J]. Molecular and Biological Evology, 25(12):2681-2688.
Jasanoff A, Wagner G, Wiley D C. 1998. Structure of a trimeric domain of the MHC class II-associated chaperonin and targeting protein Ii[J]. EMBO Journal, 17(23):6812-6818.
Koutsogiannouli E A, Moutou K A, Sarafidou T et al. 2009. Major histocompatibility complex variation at class II DQA locus in the brown hare (Lepus europaeus)[J]. Molecular Ecology, 18:4631-4649.
Kita Y F, Hosomichi K, Kohara S, et al. 2009. MHC class I A loci polymorphism and diversity in three Southeast Asian populations of cynomolgus macaque[J]. Immunogenetics, 61(9):635-648.
Lamberth K, Roeder G, Hamdahl M, et al. 2008. The peptide-binding specificity of HLA-A*3001 demonstrates membership of the HLA-A3 supertype[J]. Immunogenetics, 60(11):633-643.
Mona A S, Crestanello B, Bankhead-doronnet S, et al.2008. Disentangling the effects of recombination, selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois[J]. Molecular Ecology, 17: 4053-4067.
Rao X, Costa A I, Vanbaarle D, et al. 2009. A comparative study of HLA binding affinity and ligand diversity: implications for generating immunodominant CD8+ T cell responses[J]. Journalof Immunology, 182(3):1526-1532.
Roche P A, Cresswell P. 2011. Proteolysis of the class II-associated invariant chain generates a peptide binding site in intracellular HLA-DR molecules[J]. The Journal Immunology, 187:1076-1080.
Siebenkotten IM, Carstens C, Koch N. 1998. Identification of a sequence that mediates promiscuous binding of invariant chain to MHC class II allotypes[J]. The Journal Immunology, 160(7):3355-3362.
Tallmadge R L, Campbell J A, Miller D C, et al. 2010. Analysis of MHC class I genes across horse MHC haplotypes[J]. Immunogenetics, 62(3):159-172.
Westerdahl H, Wittzell H, Vonschantz T. 2000. Mhc diversity in two passerine birds: no evidence for a minimal essential Mhc[J]. Immunogenetics. 52(1-2):92-100.
Zhong D L, Yu W Y, Liu Y H, et al. 2004. Molecular cloning and expression of two chicken invariant chain isforms produced by alternative splicing[J]. Immunogenetics, 56: 650-656.
