| Invariant chain (Ii) is a nonpolymorphic type Ⅱ transmembrane glycoprotein, which encodes the leukocyte differentiation antigen CD74 and expresses on some cell surface. As a chaperone of MHC class II molecules, Ii plays a auxiliary effect on MHC II molecule correct folder, assembling and polymerization, and forms a nonamer with MHC II molecules into in the endoplasmic reticulum and targets the complexes to endosome body. The CLIP region (the class-II associated Ii chain peptide) of invariant chain occupies the antigen binding groove of MHC II molecules, which prevents endogenous antigen peptide from associating with the MHC II class of molecules. So Ii plays a key role in MHC class II molecule presenting exogenous antigen to CD4+ T lymphocytes and has an important regulative significance on immune reaction. Ii is currently based on DNA vaccine that has been related applications.Currently, the research of invariant chain’s structure and function focuses on the human and other mammals as well as birds, the research of fish is in the beginning stage. Ii of fish is probably the most primitive structure model. To study the structure and function of fish Ii, we cloned and sequenced first time Ii gene of grass carp by RT-PCR and RACE and analyzed bioinformatics function and structural model, while the relationship of the grass Ii and MHC quantitative expression in different tissues and the characteristics of cellular localization expression were also studied. Additionally the antibodies to grass carp invariant chain were prepared, which provided a theoretical basis for further study of structure, function and the evolutionary relationship of the invariant chain of fish as well as the relationship between the major histocompatibility complex and invariant chain.First, we successfully cloned and sequenced first time Ii gene of grass carp by RT-PCR and RACE (rapid amplification of cDNA ends). The full length Ii-cDNA was 1116 bp, its open reading frame (ORF) was 71 lbp encoding 236 amino acidsSecondly, we found that the structure of Ii of grass carp had also four domains, including transmembrane region, cytoplasm region, endoplasmic reticulum region, and a thyroglobulin (Tg) domain by comparison of grass carp Ii with the other species’Ii. The deduced amino acid sequences, the homologous and evolutionary relationships between grass carp and other different species showed that grass carp had the closest relationship with the silver carp. We further simulated the 3D structure of Ii, The comparison of them revealed that grass carp, human, mouse, chicken, goose and zebrafish were very similar in space structure, the amino acids were the same even in some improtant sites by biological information website.Thirdly, the 18sRNA gene was chosen to be a reference gene, the relationship between Ii and MHC in twelve different tissues of grass carp were detected by using the relative method of fluorescent quantitative. The results showed that a positive related in the expression of Ii and MHC in the grass carp tissue. At the same time, Ii and MHC gene were inserted into the eukaryotic expression plasmid Pmcherry-C1 and pEGFP-Nl, and further these reconstructed vectors were transfected into eukaryotic expression cell 293 T. The results showed that they located on membrane system respectively.Finally, grass carp Ii protein was obtained by prokaryotic expression and the polyclonal antibody was prepared by immunizing mice that using purified protein, we verified the specificity of the antibody by Western Blot, All of this provided a study foundation for the interaction between Ii and MHC.In all, these results demonstrated that Ii as an important immune molecule had a high degree of homology in genetics and a similarity in structure and function among different animals. The study of fish Ii has made substantial basis for the study of relation between Ii and MHC molecules. |
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