Expression And Antibody Preparetation Of Porcine IFN-γ Gene And Function Of Chicken Ii Chain

Interferon(IFN) is a kind of cytokines that have effect on anti-virus and immune system.IFN can exert antiviral activity and a number of immunomodulatory effects such as enhancement of T cell mediated cytotoxicity,B cell differentiation,MHCⅠand MHCⅡexpression,and macrophage activation.In order to research the biological function and the mechanism of the porcine interferon-gamma(poIFN-γ) in immune response,in present study this gene was cloned,expressed and generated mice anti-porcine IFN-γmulti-antibody.Firstly,poIFN-γgene was amplified from spleen lymphocytes by RT-PCR with a pair of designed primers in this experiment.The result of endonuclease TaqⅠdigesting RT-PCR products showed that the gene fragment has the same restriction enzyme site as known poIFN-γ.The PCR products was cloned into pGEX-4T-1 and pET-32a,the recombinant plasmid was identified by PCR and endonuclease EcoRⅠ+ SalⅠdigestion. The sequenc results showed that the gene of PoIFN-γconsisted of 516 nucleotides.The 0RF was consisted of 501 nucleotides,encoding 166 amino acids,molecular weight of the protein is about 17kD.The nucleotide sequence of the target gene have 100%similarity to the corresponding gene which published in Genebank.Secondly,the recombinant plasmid pGEX-poIFN-γand pET-poIFN-γwere transformed into E.coli BL21strain and highly expressed by IPTG The fusion protein solubility was identificated and the result indicated that the expressed protein located in inclusion bodies and the molecular weight were about 43.0 and 35.0 kD as we expected. After optimizing prokaryotic expression conditions,we determined the optimum inducement time and concentration of isopropylthio-β-D-galactoside(IPTG).The recombinant plasmid pGEX-poIFN-γand pET-poIFN-γwas induced to express large-scale fused protein GST-IFN-γand His-IFN-γ,the induced recombinant bacteria were lysed by freeze-thaw and sonication.At last,we obtained the GST-IFN-γand His-IFN-γinclusion body protein,which could be solubilized by sonication after the detergent lauroylsarcosine was added.Finally,mice were immunizated with the purified GST-IFN-γfusion proteins and the polyclonal antiserum against GST-IFN-γwere collected.A specific reaction appeared between the antibodies and GST-FN-γand His-IFN-γfusion protein in agar diffusion assay,ELISA and Western Blotting,these results indicated that GST-IFN-γfusion proteins can induced antibody which specificed for poIFN-γ. In conclusion,in the present study we cloned and expressed porcine IFN-γgene in prokaryocyte and induced antibody which specificed for poIFN-γsuccessfully.And this provides the further research on immunological and molecular biological properties and its amplification of recombinant poIFN-γprotein. Invariant chain(Ii) is a chaperone of MHCⅡmolecule,which mainly expresses on the surface of B cells,macrophage cells,dendritic cells and thymic epithelial cells etc.The biosynthesis of distinct forms of the invariant chain(Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice.Ii can facilitate the proper fold of the MHCⅡheterodimeric and egress from the endoplasmic reticulum.It can provide a targeting signal for endosomal/lysosomal compartments and prevents peptide from associating prematurely with MHCⅡmolecule.The endosomal sorting signal for newly synthesizedαβIi complexes were identified as two independent motifs residues at positions Leu 7/Ile 8 and Pro 15/Met 16/Leu 17 in the Ii cytoplasmic tail.Two similar signals,Leu 8/Ile 9 and Gly 16/Val 17/Leu 18,were found in the cytoplasmic tail of chicken Ii and its isoforms.To research the endosomal sorting function and its mechanism of Chicken,gene was amplified from PMD18-T/Ii-P31 by PCR using two pairs of primers,then they were inserted into pEGFP-N1 and pEGFP-C1,respectively to form two recombinant plasmids, pEGFP-N1-P31 and pEGFP-C1-P31.The two recombinant plasmids were transfected into human 293 cells strain and the cells were cultivated for 48h,then observed by a fluorescent microscope.The results show that a bright green fluorescence appeared in the endocytic compartments in the cells transfected with pEGFP-N1-P31 or pEGFP-C1-P31,but only in the nuclear in the control cells transfected with pEGFP.All these suggest that chicken Ii showed a targeting function.When mutated Leu8 or Leu 18 to Ala of chicken Ii,the GFP was still detected in intracellular compartments.While both Leu8 and Leu 18 were mutated to Ala,GFP was distributed in the cytoplasm.These results suggested that two independent endosomal sorting signal(Leu 8/Ile 9 and Gly 16/Val 17/Leu 18) were existed in the cytoplasmic tail of chicken Ii.