The Prokaryotic Expression Extracellular Structure Domain Of Sialoadhesin And Polycionai Antibody Production

The porcine reproductive and respiratory syndrome virus (PRRSV) isone of main pathogens of porcine reproductivea. The target cell of PRRSV is porcinealveolar macrophage (PAM). PRRSV infection PAM cells the depend on threereceptors: Heparin su1fate (HS),Sialoadhesin(Sn)and CD163molecules. Sialoadhesincan adsorption and mediat virus particle internalization.In this writing, Snextracellular domain structural domain recombinant protein was expressed throughprokaryotic vector and anti recombinant protein antibody was detected.Obtained PAM cells by clysis lung and extraction total RNA of PAM cells.Analysis Sn receptor extracellular domain structural domain. Amplification eightstructural domain gene fragment by RT-PCR.The size267bp、504bp、518bp、556bp、510bp、478bp、481bp、535bp.Eight gene fragment was cloned into PUCM－Tvector.Use DNAStar software analysis homology of splicing sequence with Snextracellular domain structural domain in Genbank,The result is99.1％～99.5％.We design8pairs specificity primers which5’ ends with EcoRⅠenzyme locusand3’ ends with Not enzyme locus I or. SalⅠ enzyme locus Amplification eight genefragment based on eight pairs primers. The size of them267bp、504bp、518bp、556bp、510bp、478bp、481bp、535bp,respectively.The eight nucleotide fragments wascloned into prokaryotic expression vector pET32a to constructing recombinantplasmid pET32a-Sn1, pET32a-Sn2,pET32a-Sn3. Three plasmid was expressed inIPTG induction.Obtain three protein rSn1,rSn2and rSn3and the molecular weight ofthem approximated28KDa,36KDa,37KDaconsistent with the expected proteinmolecular weight. Renaturation and purification recombinant protein by urea. Therenaturation recombinant protein was identified by SDS-PAGE electrophoresis. Thepurity of three recombinant protein85％、76％、87％. The renaturation proteinrSn1,rSn2and rSn3were inoculated mice to obtain the murine antibodies against Sn1,Sn2,Sn3recombinant fusion protein. The murine antibodies were tested by indirectELISA. The results shows that the titer of murine antibodies titer was1:12800ventually. Application of fusion protein purification by sds-page electrophoresis andWestern blotting exprement use specificity polyclonal antibody.The result show thatspecificity polyclonal antibody could binding their recombinant protein,identification that recombinant protein have good immunogenicity.