The Enhancement Of Bovine Prolactin Signal Peptide On The Expression Of Recombinant Porcine Circovirus 3 Capsid Protein With More Neutralizing Epitopes

Porcine circovirus 3(PCV3)is one of the smallest animal viruses.PCV3discovered in the past two years is a pathogen that induces porcine dermatitis nephrotic syndrome and reproductive disorders.Infection with PCV3 will cause porcine dermatitis and nephropathy syndrome(PDNS),multisystemic inflammation,and reproductive failure.However,the current research on commercialized vaccines for PCV3 is still a vacancy.Capsid protein(CP)is the only structural protein of PCV3,and it is also the main antigen protein of PCV3.We predicted three neutralizing epitopes of PCV3 by analyzing the hydrophilicity,fold-ability,surface accessibility and secondary structure of PCV3 CP(epitope A、epitope B and epitope C).The decoy epitope of PCV3 CP has not been reported so far,while the decoy epitope of PCV2 CP has been determined.Because of the homology of PCV2 CP and PCV3 CP,we believe that PCV3 CP also has a decoy epitope.We found the conserved sequence of PCV3 CP and PCV2 CP through sequence alignment between PCV2 CP and PCV3 CP.Since the decoy epitope of PCV2 CP is just behind this conserved sequence,we predicted the decoy epitope(epitope D)of PCV3 CP is also behind this conserved sequence.Neutralizing epitopes induce the production of neutralizing antibodies that neutralize viral antigens while decoy epitopes induce immune escape.In order to obtain PCV3 recombinant capsid protein capable of inducing more neutralizing antibodies,we replaced epitope D with epitope C on the basis of PCV3 CP,namely recombinant porcine circovirus 3 capsid protein with more neutralizing epitopes(protein X).We synthesized the coding gene of X protein by PCR,and then constructed the recombinant plasmid pET28a-X,X protein was expressed in E.coli in the end.The results showed that the expression of X protein was very low.In order to increase the expression of recombinant PCV3 capsid protein with multiple neutralizing epitopes,we have noticed that the partial deletion of nuclear localization signal region and the fusion of signal peptide could enhance the protein expression.Arg-rich peptide in the nuclear localization sequence(NLS)reduces the expression of recombinant PCV2 capsid protein.It has been reported that fusion of bovine prolactin signal peptide(BSP)can enhance the expression of PCV2 capsid protein in an eukaryotic system.Based on the homology between PCV3 CP and PCV2CP,we assume that deleting NLS or fusing BSP could increase the expression of recombinant PCV3 capsid protein in E.coli.Summing up the above analysis,we constructed a 5R~—multi neutralizing epitope recombinant PCV3 capsid protein(XM protein)by deleting five consecutive arginine from NLS,and we constructed a multi neutralizing epitope recombinant PCV3 capsid protein(BSP-X protein)fused with BSP on the basis of protein X,we finally expressed them in E.coli.The results showed that the expression of protein XM did not increase compared with X protein,but protein BSP-X was highly expressed.The result indicated that BSP could enhance the expression of recombinant PCV3 CP with more neutralizing epitopes.We purified protein BSP-X by ammonium sulfate precipitation.We immunized mice with purified BSP-X protein,detected the antibody level in the serums and the activation of the immune cells in the spleens.The results showed that BSP-X protein could induce high antibody level.BSP-X could stimulate the proliferation and activation of T cells and B cell in mice.In summary,bovine prolactin signal peptide can enhance the expression of recombinant porcine circovirus 3 capsid protein with more neutralizing epitopes,and BSP-X has strong immunogenicity.