Epitope Prediction And Recombinant Expression Of H5N1 Avian Influenza Virus Hemagglutinin Protein

S-layer protein CTC surface display system of Bacillus thuringiensis was used to display avian influenza virus hemagglutinin HA1 protein on the cell surface of Bacillus thuringiensis by Liu Mei(2007) in our laboratory,and the specific antibodies against AIV H5N1 subtype were producted after chickens were immunized with recombinant strains.But by increasing the immunization dose and frequency,the specific antibody titer was only up to 3.8log2,which was difficult to achieve the immune effect in practical application.The reason could be that the expression volume was too little or the configuration of the antigenic sites expressed did not meet the optimal levels.In this study,epitopes of the HA1 protein of the H5N1 avian influenza virus were predicted by biology software.Then the epitopes of different configurations were cloned and expressed in the Bacillus thuringiensis S-layer protein.This study was the foundation for the development of the heat-stable and highly efficient genetic engineering vaccine about avian influenza.1.The prediction of the HA1 protein epitopes of H5N1 avian influenza virusThe secondary structure of the HA1 protein of the H5N1 avian influenza virus was carried out by centralα-helix,β-folded area,corner,curl-free rules,flexibility, hydrophilicity,surface probability and antigenic index through the use of SignalP3.0 sever,DNAStar software,and so on.The six objective fragments called A,B,C,D,E and F were achieved after comprehensive analysis.2.Cloning of the HA1 protein epitopes of H5N1 avian influenza virusPrimer 5.0 software was used to design six pairs of primers,and pKG-HA1 and pKG-HA1M were used as templates of PCR in order to get the target fragments.Then, the target fragments were cloned into pGM-T vector to get the positive clones of pGM-A, pGM-B,pGM-C,pGM-D,pGM-E and pGM-F.The results showed that,the sequences of PCR products were in line with the original sequence,and the restriction sites were in line with the designed ones.The target fragments were connected to the plasmid pBMB982-304 relatively by use of the restriction sites of XbaⅠand HincⅡ,which lead to the recombinant plasmids pCTC-A,pCTC-B,pCTC-C,pCTC-D,pCTC-E and pCTC-F.Using EcoRⅠsite,csaAB operon was inserted in pCTC-A,pCTC-B and pCTC-E to get new recombinant plasmids pCSHA1A,pCSHA1B and pCSHA1E. 3.Construction of recombinant strains which harboring target genes and S-layer fusion genesNine recombinant B.thuringiensis strains were constructed by transferring recombinant plasmids to receptor BMB171.The resulting strains included BCA (harboring pCSHA1A),BCB(harboring pCSHA1B),BCE(harboring pCSHA1E), BCCA(harboring pCTC-A and pMIL-CSA).BCCB(harboring pCTC-B and pMIL-CSA),BCCC(harboring pCTC-C and pMIL-CSA),BCCD(harboring pCTC-D and pMIL-CSA),BCCE(harboring pCTC-E and pMIL-CSA) and BCCF(harboring pCTC-F and pMIL-CSA).4.Assaying the display of exogenous protein in recombinant strainsHemagglutination assay showed that some recombinant proteins were displayed on the cell surface of respective recombinant strains.Hemagglutination inhibition assay showed recombinant proteins displayed on the cell surface of recombinant strains respectively were specific to standard positive serum of avian influenza virus H5N1.