Fine Mapping Of Linear B-cell Epitopes Of Peste Des Petits Ruminants Virus Hemagglutinin Protein

Peste des Petits Ruminants?PPR?is caused by the Peste des Petits Ruminants Virus?PPRV?,which is an acute contagious disease.Hemagglutinin protein?H protein?binds to its cognate receptor on the host cell and confers highly protective immunity.In this study,the modified biosynthetic peptide method was employed to mapping the linear B cell epitopes on PPRV H protein by western blot using anti-recombinant PPRV H protein antiserum from rabbits as the primary antibody.The following experiments was carried out in this project:1.Screening of 16 peptide fragments containing linear B cell epitopesFirstly,overlapping DNA fragments encoding 16-mer peptides covering the full length of the H protein were designed and synthesized.BamH ? and TAA-Sal ? were added to the 5' and 3' ends of each DNA fragment,respectively.Then the annealed DNA fragments were ligated into prokaryotic expression vector pXXGST-3 to obtain the corresponding 16 peptide expression vector p XXGST-3-P75-?1 75?.The sequencing confirmed recombinant plasmids were transform into E.coli BL 21 competent cells and the positive clones were selected by SDS-PAGE.The positive 16 peptide fragment containing BCE was screened by Western blot,and 14 positive 16 peptides were obtained from 75 GST fusion expression 16,i.e.P1,P9,P10,P11,P13,P28,P45,P46,P47,P48,P58,P60,P63 and P74.2.Identification BCE minimal motifs of the positive 16 peptidesSecondly,DNA fragments encoding series of 8 peptides covering the full length of each positive 16 peptide and overlapping each other with 7 a mino acid residues were designed and synthesized.BamH ? and TAA-Sal ? were added to the 5 'and 3' ends of each DNA fragment synthesized,respectively.Then the in vitro annealled DNA fragments were ligated into p XXGST-3 to obtain the 8 peptide expression vectors.The sequences of plasmids were confirmed and then transformed into E.coli BL 21 competent cells and the positive clones was selected by SDS-PAGE.The minimal epitope motif of each positive reaction 16 peptide was identified by Western blot using anti-recombinant H protein serum as the primary antibody.The results show ed that 1-6 positive 8 peptides were identifed in the series of 8 peptides corresponding to each 16 positive peptides except P13,all the 8 peptides corresponding to which are negative.The minimal motifs identified are: ⁸INA¹⁰?In P11?,²²⁴EEGLFGRT²³¹?in P28?,³⁵⁹KDDEANW³⁶⁵?in P45?,³⁶⁷VPSTDV³⁷²?in P46?,and the total number of B-cell epitopes were obtained: ⁸INA¹⁰?in P1?,⁷³RLNTNIKL⁸⁰?in P10?,⁸⁴IDHQT⁸⁸?in P11?,³⁷²VRDLQ³⁷⁶?in P47?,³⁸³VEACKTR³⁸⁹?in P48?,⁴⁶³FLGMINT⁴⁷⁰?in P58?,⁴⁷⁸VMPHILT⁴⁸⁴?in P60?,⁵⁰⁴VDDDIK⁵⁰⁹?in P63?and 590TDEEVHT⁵⁹⁶?in P74?.3.Identification of the minimal motif of positive 16 peptide P13.Since western blot results of series of 8 peptide corresponds to the positive 16 peptide P13 were all negative,we used substracing peptides method to identify the minimal epitope motif.DNA fragments encoding two series of 15-9 peptides minus 1-7 amino acid residues from the N-terminus or C-terminus were designed and synthesized.the in vitro annealed DNA fragments were ligated to the p XXGST-3 to obtain series of recombinant 9-15 peptide GST fusion express plasmids.Sequencing confirmed recombinant plasmids were transformed into E.coli BL 21 competent cells and the positive clones were identified by SDS-PAGE.The minimal epitope motif of P13 was identified by Western blot using anti-recombinant H rabbit serum as primary antibody.A 10-mer minimal epitope motif was obtained: ⁹⁸?GDEVGIRI¹⁰⁷.4.Reactivity of the identified BCEs with serum from goat immunized with vacci ne strain PPRVThe reactivity of identified BCEs were tested by western blot using serum from a goat immunized with vaccine strain PPRV 75/1 as primary antibody.The results indicated that nine out of the 13 BCE peptides containing one minimal motif identified each reacted with the goat serum.5.3D structure modeling of the minimal motifs of the identified BCEsTo clarify the spatial locations of the 13 minimal motifs of the BCEs identified on PPRV H protein,we generated 3-dimensional structural models of PPRV H protein using intensive modeling by Phyre2 server.The predication results showed that 412 of 471 residues?89%?of the head domain of H were modeled at >90% confidence;the remaining 59 residues were modeled by ab initio.However,none of residues of the cytoplamic tail and stalk region was modelled at >90% confidence.The mapped BCE motifs were distributed at the regions of predicted loops,?-sheets and helixes,and ten of 13 identified BCE motifs were exposed on the surface of the predicted 3D structure,suggesting that they all may be antibody-accessible BCE motifs.The other 3 BCE motifs?epitope motifs 5,7,10?almost entirely buried inside the PPRV H protein.