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Molecular Biological Characteristics of Multiple Porcine Fc Gamma RⅡB Sub-isoforms Generated by Alternative Splicing

Author: LiuXiaoPing
Tutor: XiaPingAn;CuiBaoAn
School: Henan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: porcine FcγRⅡB splicing isomer gene cloning eukaryotic expression cytokine
CLC: S828
Type: Master's thesis
Year: 2011
Downloads: 5
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Abstract


FcγRⅡB plays an important role on the aspect of negative regulation in the immune response of innate immunity and adaptive immunity,which is found as the only one inhibitory Fc receptor.This study cloned and determined several FcγRⅡB transcripts of porcine,and it is significant to further investigate the biological characteristics of FcγRⅡB thorough and the mechanism of immune regulation that FcγRⅡB mediated with.The gene of FcγRⅡB was cloned from porcine pulmonary alveolar macrophages into six transcripts by RT-PCR,and theORF sizes are 951,891,894,948,639and 519bp respectively. Analysis of sequences and structures showed that The gene sequence of transcript 1 codes 316 amino acids in all,which contain N-terminal signal peptide of 45 amino acids, extracellular domain of 179 amino acids,which include two Ig structural domain,EC1 and EC2, hydrophobic transmembrane area of 23 amino acids and intracellular region of 69 amino acids.We analyzed the sequences of the other five transcripts based on the amino acids sequence of transcript 1.And we find that the N-terminal of transcript 2 and transcript 4 are lack of a alanine,and its signal peptide only constitute 42 amino acids.The transcript 2 and transcript 3 are lack of 19 amino acids on the intracellular region. The transcript 5 is lack of EC2 in the extracellular domain and 19 amino acids in the intracellular region. The transcript 6 is lack of EC2 of extracellular domain and transmembrane area and 27 amino acids of cytoplasmic tail area,and it’s predicted as a derivation of the soluble Fc receptor.The transcript 1, transcript 2 and FcγRⅡB(FJ608551、DQ026064),which have been identified before, are homologous.The transcript 2, transcript 4, transcript 5 and transcript 6 are four new FcγRⅡB of porcine.At present.in humans and other animals,we have not yet found FcγRⅡB ,which structure is similar as transcript 5 and transcript 6. The six transcripts compared with DQ026064 sequence,Homology of nucleotide sequence of porcine six FcγRⅡB transcript variants were 98.8%, 99.1%, 99.6%, 98.2%, 98.7%, 98.3% ,and amino acid sequences were 97.0%, 97.6%, 98.3%, 96.3%, 96.7%, 95.4% homology.Analyzing FcγRⅡB gene sequence,we found that six transcripts resulted from the alternative splicing of mRNA ,and all belonged to the alternative splicing isoforms, and they are FcγRⅡb 1, FcγRⅡb2, FcγRⅡb3, FcγRⅡb 4, FcγRⅡb5 and FcγRⅡb6 .The FcγRⅡb1-6’ ORF genes were subcloned into eukaryotic expression vector pcDNA3.0 to construct the expressing plasmids and then transfected into COS-7 cells with liposomes.The transfected cells were treated with mouse anti-pig FcγRⅡB positive serum and negative serum respectively.After cells detected by flow cytometry,the data of FcγRⅡb1-6 groups treated with positive serum,empty vector of pcDNA3.0 transfected and untransfected COS cell lines were 25.3%,17.7%,20.0%,18.3%,14.4%,2.3%,2.5%,3.0%,and the negative serum groups were 2.7%,2.0%,2.2%,1.3%,1.5%,1.5%,1.3%,1.8%.The results showed that the expression of receptor protein was detected from the cell surface of FcγRⅡb 1-5 transfected cell lines up to 10% and could not be detected from FcγRⅡb6, empty vector transfected cell lines and untransfected cells. So,FcγRⅡb1-5 were successfully expressed on the transfected cells’ surface.The cells transfected with FcγRⅡb1-6 eukaryotic expression vector were selectively cultured 15 days with 400μg/mL G418 after transfection 24h and detected by indirect immunofluorescence. The results showed that the FcγRⅡb1-5 transfected cells fluorescence detection rate were all above 80%.But transfected with FcγRⅡb6 and empty vector cells and untransfected COS7 cells have not been observed under fluorescence. It showed that we have established stable expression of FcγRⅡb1-5 receptor protein on COS-7 cell lines initially.Biological activity of the splicing isomers of porcine FcγRⅡB binding with immune complex was tested by rosette test and the result shows that surrounding of transfected cells with FcγRⅡb 1-4 forms obvious rosette,and the surroundings of cells transfected with FcγRⅡb 5、FcγRⅡb 6、pcDNA3.0 and untransfected cells did not appearing rosette.The results indicate that porcine FcγRⅡb 1-4 have the biological activity of binding with IgG immune complex,but FcγRⅡb5 does not have it.Five times diluted mouse anti-pig FcγRⅡB extracellular region serum were used to close PAM cells, and then the non-infectious IgG immune complex with different concentration treated PAM cells which stimulated by LPS. Relative real-time PCR method was used to detect IL-10, IFN-αand GM-CSF transcription at 6h,12h,18h,24h. The results showed that IL-10、IFN-αand GM-CSF transcriptional level up-regulation obviously after serum blocking. The results indicated that IgG immune complex could inhibit IL-10, IFN-αand GM-CSF transcription through FcγRⅡB.

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