Identification Of Linear B Cell Epitopes In S1^0A Region Of PEDV S Protein

PEDV can induce porcine epidemic diarrhea（PED）,an acute and highly infectious intestinal disease in pigs.In 2010,there was a large outbreak of PED in China.At this time,PEDV was mutated,leading to the previous vaccine immunization failure.S protein is a punctured protein on the surface of PEDV particles,which plays a very important role in invading pig intestinal cells.Researchers have found that S protein can induce infected hosts to produce protective neutralizing antibodies against PEDV.The researchers compared the gene sequences of multiple PEDV strains and found significant differences in the S gene.There was a highly variable region between amino acid sequences 1 to 496 at the N-terminal of the S protein,named S1^0A.Identification and conservative analysis of the B cell Epitope mapping in the PEDV S1^0Aregion were helpful to analyze the immune escape mechanism of PEDV variant strains at the Epitope level,and also provide a basis for the preparation of Epitope vaccine.In this study,the biosynthesis peptide method was used to identify the linear B cell epitopes in the highly variable region of PEDV S protein using the rabbit serum retained in the laboratory as the primary antibody.The main studies are as follows:1.Prediction of linear B cell epitopeson PEDVS1^0AfragmentThe S protein of PEDV CV777 vaccine strain was selected as the research target.Considering the hydrophilicity index,flexibility,protein surface accessibility and antigenicity index of the amino acid residues contained in S1^0A,the Protean module in DNAStar software was used to analyze and predict the linear B cell epitope.Combined with the spatial structure simulated by Phyre2 online tool,four linear B cell epitopes were predicted on PEDV S1^0Afragment:⁹⁴EPFDPSG¹⁰⁰,¹⁰⁷KATNGN¹¹²,¹²⁵PDNKTLG¹³¹and ^{3 73}VDTYKST³⁷⁹.Objective to find out the precise target of universal antibody and provide the design direction for epitope vaccine.2.Screening of PEDV positive porcine serumTo acquire positive porcine serum for the screen linear eipitope on PEDV S protein,the previous prepared recombinant expression plasmids p ET-32a-CF₁,p ET-32a-CF₂,and p ET-28a-CF₃,respectly carrying optimized 3 fragments of S gene,were transformed into E.coli BL21competent cells,and the expression of three recombinant proteins were induced by IPTG.More than 20 porcine-anti-PEDV sera keped in our laboratory were used as the primary antibody to screen e porcine sera reacting with the three recombinant proteins by western blot.The results showed that there were positive reactions between 6^#pig serum and three segment recombinant protein,which provided an important basis for antibody accessibility verification3.Identificationof minimal B cell epitope motif on PEDV S10AThe S1^0Apositive 16 peptide was synthesized into 9 8 peptide encoding DNA sequence which overlapped with 7 residues.Bam H I restriction site was added to the 5’-end and Taa-Sal I restriction site was added to the 3’-end.p XXGST-3-CV777（38-62）recombinant 8 peptide expression vector was obtained by ligating the recovered p XXGST-3vector to synthesize 8 peptide.More than 100 8-peptide vectors were constructed in the experiment,which laid a foundation for the identification of the minimum cell epitope motif of PEDV S1^0Ain the future.4.Identification of linear epitopes of two B cells on PEDV S proteinIn this study,rabbit anti-PEDV（CF₁）and rabbit anti-PEDV（CF₃）were used as primary antibodies and HRP anti-rabbit as secondary antibodies.Two linear epitopes of B cells,⁶³CGTGIETDSGV⁷³and¹³⁶⁸PRLQPYEAFEK¹³⁷⁸,were screened out by western bolt to provide basis for making epitope vaccines in the future.