Cloning And Characterization Of Porcine Fc Gamma Receptor Iib1 Transcript Variant

In PRRS studied to date,the process of infection for PRRSV existed the antibody -dependent enhancement phenomenon, the PRRSV infectted target cell which was enhanced by the link of deuto-neutralization dose antibodies and Fc receptors.Four different classes of Fc receptors have been defined:FcγRI(CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV. FcgRII (CD32) is an FγR previously shown to exist three isoforms: FcγRIIA,FcγRIIB and FcγRIIC. FcγRIIB is an inhibitory FγR, Two transcript variants of FcγRIIB have been demonstrated in humans and mice, In this study,we cloned and characterizated swFcγRⅡB transcript variant,whitch established foundation to study and research the process of infection for PRRSV.According to the cDNA sequence of the porcine Fc gamma receptor IIB (swFcγRIIB ) in the GeneBank,a pair of primer was designed using primer.The gene of swFcγRⅡB transcript variant was amplificated from porcine peripheral white blood cells using RT-PCR. Amplificated gene fragment was inseted into pTG19-T vector to sequencing.The putative epitope of the swFcγRⅡB transcript variant's amino acid sequence was predicted by DNAStar Protean.According to the seqense analysis,the swFcγRⅡB transcript variant's cloning gene fragment is 951bp,and encodes 316 amino acids, The first 45 amino acids were predicted to be an N-teminal secretory signal peptide.The mature protein is composed of a 179 amino acid extracellular region followed by a hydrophobic region of 23 amino acids representing a putative trans-membranedomain and a 69 amino acid cytoplasmic tail. The extracellular region was found to include five potential N-glycosylation sites (Asn 34, Asn 44, Asn 135 Asn 142 and Asn 166) and four conserved cysteines that form the characteristic disulphide bonds of the immunoglobulin domains. From the conserved immunoglobulin domain structure it is suggested that Cys 71 is linked to Cys 113 to formdomain 1 (EC1), and that Cys 152 is paired with Cys 192 to form domain 2 (EC2).The homology of nucleotide and amino acid sequence between FJ608551 and DQ026064 is 99.3% and 98.3%.Its intracellular region is augmented 19 amino acids compared with the publicated sequence.Its amino acid sequence exists more than 3 putative dominant antigen epitopes. The porcine Fc gamma receptor IIB exists subgroup,the gene we got is one kind of RNA transcript variant of the porcine Fc gamma receptor IIB.It can be predicted that the RNA transcript variant of the porcine Fc gamma receptor IIB exists at least two kinds. The swFcγRⅡB transcript variant cDNA codes for a cytoplasmic amino acid sequence of 69 amino acids and so appears most similar to the FcγRIIb1 isoform in human.According to the cDNA sequence(FJ608551)of the porcine Fc gamma receptor IIB's (swFcγRIIB's)RNA transcript variant in the GeneBank, four pair of primers were designed using primer. The gene was amplificated from swFcγRⅡB transcript variant abscised intracellular region and transmembrane domain,extracellular region,EC1 region of swFcγRⅡB transcript variant, EC2 region of swFcγRⅡB transcript variant.The PCR products were cutted by kpnI and EcoRI, The swFcγRⅡB transcript variant truncated gene were cloned into pET-32a vector to generate the recombinant expressing plasmid pET-ZY,pET-EY,pET-AY,pET-BY.The swFcγRIIB -His fusion protein was successfully obtained with IPTG, The protein predominantly existed as inclusion bodies in the cytoplasm.After expression in E.coil and purification ,the purifide protein swFcγRIIb1-ZY, swFcγRIIb1-EY, swFcγRIIb1-AY, swFcγRIIb1-BY were used to immunizi mouse to obtain the antiserum. The results of ELISA and Western blotting indicated that the prepared ployconal antibody had high titer and specificity. The purifide protein of swFcγRIIB-His and its polyclonal antibody lays the foundation for fourther reserch on swFcγRⅡB transcript variant.According to the cDNA sequence(FJ608551)of the porcine Fc gamma receptor IIB's (swFcγRIIB's)RNA transcript variant in the GeneBank, a pair of primer was designed using primer. The gene was amplificated from the complete swFcγRⅡB transcript variant. The PCR product was cutted by kpnI and EcoRI, The swFcγRⅡB transcript variant gene was cloned into pcDNA3.0 vector to generate the recombinant expressing plasmid pcDNA-swFcγRIIb1.Thongh double digestion by kpnI/EcoR I and PCR identification,951 bp of DNA fragment was obtained,indicating that the recombinant plasmid pcDNA-swFcγRIIb1 was successfully constucted .The 951 bp fragment was then transfected into COS-7 cells by the liposome method, Flow-cytometric and Immunofluorescence detection analysis expression of the swFcγRⅡB transcript variant proteins on COS-7 cells,and the ligand affinity of was identified by Rosetting procedures.