Cloning, Prokarytic Expression And Immune Reactivity Of Chicken NDV-F And Eukaryotic Synchronous Expression Of Chicken Ii And MHC â…¡

Newcastle disease is a septic infection and pestilent disease,which is caused by Newcastle disease virus and be called Philippine fowl disease also.Symptoms of Newcastle disease include hemorrhage from respiratory tract and alimentary tract.Office Internationaldes Epizooties classify Newcastle disease as A grade,which severely impair animal husbandry and human health.In recent years,ND had broken out many times,in China,which not only intimidate broiler-fryer industry,also caused huge economic losses in poultry industry.So the prevention from the disease of NDV has been in a unprecedent way put forward in the public hygiene field and it is very urgent to find a perfect protective antigen,which is used to diagnose and prevent this disease now.Fusion Protein(F)is a major surface glycoprotein of NDV,which plays a key role in entering the cytoplasmic membrane of host target cells,which is bound up with the viral pathogenicity and the toxicity.First,Fusion Protein is synthesised by the form of F0,which has no activity.When F0 is transported to the surface of golgi membrane,it will be break into the form of F1 and F2 by protease of the host cells,which makes virus owning infectious activity.When the unit of enzyme activity just catches the surface of the pericellular membrane,it will makes the infected cell to graft the clean cell,and to extend period of infection progressively.It is of great importance to study F protein to reveal the mechanism of NDV and manufacture NDV vaccine with antiadsorption,which is one of the focus in the research of F immunoprevention now.In order to reveal the Fusion Protein's constitutional features,inquire into the structure of F protein and obtain abundant recombinant F protein,which would supply the basis to further prepare the McAb of NDV, research its function and prepare F vaccine,in this study F gene of chicken was cloned and sequenced,recombinant plasmids pGEX-4T-1-F were construct and prokaryotic expressed.Firstly,according to mRNA of NDV(F48E9)F published by GenBank,a pair of primers,was designed.F gene of NDV(F48E9)was amplified by PCR from the recombinant plasmids pcDNA-F,it was 594bp in length.The results of endonuclease FbaI digesting PCR products,showed that the gene fragment have the same restriction enzyme sites as known NDV(F48E9)F.Secondly,The PCR product was cloned into prokaryotic expressive vector pGEX-4T-1.The recombinant plasmids were identified by PCR and endonuclease EcoRⅠ+ SalⅠdigestion.The recombinant,namely pGEX-4T-1-F,was transformed into E.coli strain BL21 and GST-F fusion proteins were induced to express.The MW of the fusion proteins were about 46 000 as analyzed by SDS-PAGE,respectively.And the fusion protein's solubility was identified,the expressed fusion protein GST-F located in inclusion bodies.Finally,after optimizing prokaryotic expression conditions,we determined the optimum inducement time and concentration of isopropylthio-β-D- galactoside(IPTG). The recombinant plasmids pGEX-4T-1-F was induced to express large-scale fusion protein GST-F,the induced recombinant bacteria were lysed by freeze-thaw and sonication.At last, we obtained the GST-F inclusion body protein,which could be solubilized by sonication after the detergent lauroylsarcosine was added.Western-blot analysis with antibodies against NDV showed that the recombinant protein has good antigencity.In conclusion,we cloned and expressed F gene in prokaryocyte successfully.All these can provide a solid foundation for preparing NDV McAb and studying the function of F protein,also for further developing the NDV vaccine. ClassⅡmolecules are glycoproteins that are formed by two noncovalence-associated chainsαandβ,each of which is encoded by different MHC genes with extraordinary polymorphism.ClassⅡmolecules have an effect on presenting antigen and activating T cells by forming classⅡmolecule-antigen peptide complex,which can stimulate T cells and B cells to participate in immune response.The invariant chain(Ii)is a non-polymorphic typeⅡtransmembrane protein and plays a central role in regulating the expression and funstocompatibility complex(MHC)molecules.Firstly,according to mRNA of MHCⅡ-α/βand Ii gene sequence registered in GenBank and multi-cloning restriction enzyme sites of vectorction of classⅡmajor hi pEGFP-C1 and pDsRed2-N1,three pairs of specific primers were designed.Three gene fragments which encode MHCⅡ-α,MHCⅡ-βand Ii were amplified from recombinant plasmids pGEX-4T-1/α,pGEX-4T-1/β,pcDNA3-Ii by PeR,they were 771bp,798bp and 672bp in length,respectively,gene fragments ofα,β,Ii were ligated to pEGFP-C1 vector to construct the eukaryotic expressive recombinant plasmids GFP-α,GFP-βand GFP-Ii respectively.Then Ii was ligated to pDsRed2-N1 vector to construct the eukaryotic expressive recombinant plasmid RFP-Ii.The recombinants were analysed after they were identified by PeR and endonuclease BglⅡ+ SalⅠ/EcoRⅠ+ SalⅠ/EcoRⅠ+SalⅠ/ EcoRⅠ+SalⅠdigestion respectively.Secondly,Chicken MHC classⅡmolecules and Ii show high similarity to their mammalian homologues at amino acid level,they were supposed to play important roles in foreign antigen presentation as mammalian homologues do.There have no reports on interaction between MHC classⅡmolecules and Ii in chicken.We co-transfected GFP-α, GFP-βand Ii gene into 293 cells,co-localization of three proteins were observed in (immune)fluorescence microscopy.The result indicated that interaction between chicken MHC classⅡmolecules and Ii might exist.Finally,We co-transfected GFP-α,GFP-βand Ii gene into 293 cells,or transfected GFP-α,GFP-βand Ii gene into 293 cells respectively.Recombinant proteins were obtained after 36h.We study the correlation between the MHCⅡand Ii by Western-blot.The result of Western-blot showed that each of the molecular weight of GFP-α,GFP-βand Ii of co-transfection has changed.The cause that the molecular weight is change is not still clear.In conclusion,we constructed recombinant eukaryotic plasmids GFP-α,GFP-β,GFP-Ii and RFP-Ii successfully,observed three proteins in fluorescence microscopy.The result indicated that interaction between chicken MHC classⅡmolecules and Ii might exist. All these can provide a solid foundation for studying the correlation between the MHCⅡand Ii,also for further developing the Ii vaccine.