Studies On The Cross-Reactive Immunity And Functional Fc-peptides Of IgG Antibody For Raccoon Dog

As a molecule, IgG functionally combines facets of both innate and adaptive immunity and combines antigen binding sites with binding sites for many innate receptors and adaptor molecules as a bridge. The multifunction immunoglobulin IgG belong to mammals in vivo is also the main antibody when immune responses again.IgG bind to phagocytic cells and NK cell which has Fc?R through Fc fragment. This response mediated by a series of biological effects, including neutralizing toxins,affinity and cause cell to gobble up regulate the role, extracellular killer and immune inflammation, ADCC effect, activate complement way, etc. The antigenicity of antibody itself mainly depends on the Fc period of antibody's heavy chain. Encoding a species IgG gene of the heavy chain constant region is relatively conserved, so the analysis of different animal's homologous of IgG-Fc gene itself may reflect the different animal antibody presence antigenic cross. The test selected racoon dog IgG gene for the study.This research consists of three parts as follows:Part ?, The gene sequencing and sequence analysis of racoon dog IgG heavy chainA pair of primer was designed according to the conserved domain of dog and mink's IgG heavy chain published in the NCBI, viral RNA was extracted from the sick racoon dog' spleen. Using the conserved domain and 3'Race to amplification complete sequence, and the length of the complete sequence was 1474 bp, coding 491 amino acid. Different kinds of software to analyze the bioinformatics and genetic evolution of IgG intensively.The result showed that affinity between racoon dog and dog were closest, the homology of nucleotide sequence was 92.6%, the homology of racoon dog and cat was 83.0%, the homology of racoon dog and mink was 82.8%,the homology of racoon dog and human was 75.9%, the homology of racoon dog and mice was 68.2%. The homology of amino acid sequence was 89..6%, the homology of racoon dog and mink was77.2%,the homology of racoon dog and human was about70%, the homology of racoon dog and mice was about 60%.Through the homology modeling of the Swiss-model and the three-dimensional crystal structural prediction of a variety of animal IgG of Fc, however, IgG class based on human IgG-Fc and three-dimensional structural similarity are about 70%.The difference of the Fc binding sites and three dimensional space structure is genetic variation of the IgG- Fc.Part ?, Cross-reactive immunity analysis of different species of animals' IgGThree-dimensional structures of different IgG-Fc molecules have a higher rate of overlap, moreover,the homology of amino acid residues in Fc region binding among different animals have differences to different extent,so these suggest that different animals' IgG can cross reaction with anti-antibody. By expressing racoon dog IgG-Fc gene and using animals' serum antibody and HRP-IgG,dot-ELISA and Western-Blot test were done. It was tested the cross reaction of anti-dog IgG,anti-rabbit IgG,anti-human IgG and anti-mouse IgG as anti-antibodies to mice, chicken, duck,dog,racoon dog,human,rabbit, mink's IgG. The results showed that there was more or less some cross between different animals' IgG. Animals between different schema may also have cross-immune response, such as a significant cross-reactivity between the chicken and the dog, compared to the rabbit, mouse and human.Part ?, Functional analysis of Fc-peptides of IgG for raccoon dogTo date, there is no report about functional peptides of IgG antibody in racoon dog.short synthetic peptides N10K(NTVSITCLVK) or P10Q(PSVYVLPPSQ)corresponding to sequence of IgG-Fc region of racoon dog have been shown that they can stimulate in vitro the production of proinflammatory cytokines( TNF-?,IL-1?,IL-6) in peripheral lymphocytes of mice to a certain degree by ELISA. Fc-peptide N10 K can induce more production of TNF-?(271.95pg/m L),IL-1?(27.42pg/m L), while the peptide P10 Q can stimulate mouse peripheral blood lymphocytes to secret more IL-6(35.51pg/m L). To our knowledge, it is the first to report the characteristics of IgG antibody of racoon dog, the study improve our understanding of racoon dog IgG antibody and provide the theoretical foundation for therapeutic antibody development and the research of immunology.