Construction And Antigenicity Of Recombinant Phage Expression Of Neutralizing Epitopes Of GP5 Protein From Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome?PRRS?is a highly contagious disease which caused by Porcine reproductive and respiratory syndrome virus.So far,the disease has been mainly treated with preventive measures,and vaccination is the primary measure to prevent the disease.GP5 protein is an important structural protein of PRRSV,and there are six important antigenic determinants,which can induce the production of neutralizing antibodies,so it has been an important protein for vaccine development.Among them,the neutralizing B epitope has been confirmed as the neutralizing antigen region of the virus.This study aimed to investigate the neutralizing antibodies produced by neutralizing B cell epitopes and their effects.A total of 430 positive field serums from 7 farms vaccined with different commercial vaccines respectively were tested by epitope B based indirect ELISA.The results showed that the percentage of positive serum antibody levels in different farms ranged from 86.11%to 98.15%?Using the neutralizing epitope B epitope as the target sequence,fuse with the targeting peptide DC3pep and the non-targeting peptide SCRMpep respectively,then construct the recombinant phage M13-DC3pep-GP5-B and M13-SCRMpep-GP5 by phage display technology,use clinical positive serum for western blot analysis of the immunogenicity of both.Mice were immunized with1.0×10¹³pfu/mL recombinant phage M13-DC3pep-GP5-B,M13-DC3pep-GP5-B mixed Freund's adjuvant and M13-SCRMpep-GP5,then specific antibodies were detected of mouse serum;The results showed that the both the recombinant phage M13-DC3pep-GP5-B and M13-SCRMpep-GP5-B have a good reactogenicity.The mice immunized of M13-DC3pep-GP5-B mixed Freund's adjuvant produced specific antibody,antibody levels reached a maximum of 0.94 in the fourth week;In the immunized group of M13-DC3pep-GP5-B,the antibody level of mice reached a maximum of 0.93 in the sixth week,In the immunized group of M13-SCRMpep-GP5-B,the antibody level of mice reached a maximum of 0.634 in the sixth week.The specific antibody produced by recombinant phage M13-DC3pep-GP5-B was higher than immunized by M13-SCRMpep-GP5-B,and the difference of them was significant?p<0.05?.The weaned piglets were immunized with recombinant phage M13-DC3pep-GP5-B?M13-DC3pep-GP5-B mixed aluminum hydroxide adjuvant and M13-SCRMpep-GP5-B at a dose of 1.0×10¹³ pfu/mL.The specific antibodies of piglet serum were detected,the neutralization test of piglets were tested by using clinical isolation of PRRSV strains;The results showed that the antibody level of piglets immunized M13-DC3pep-GP5-B mixed aluminum hydroxide adjuvant reached the highest level of 0.687 in the fourth week after the last immunization,and its antibody level was significantly higher than piglets immunized M13-DC3pep-GP5-B group and M13-SCRMpep-GP5-B group?p<0.05?;the specific antibody level of piglets immunized by M13-DC3pep-GP5-B was higher than that piglets immunized by M13-SCRMpep-GP5-B,the difference was not significant?P>0.05?;The results showed that the neutralizing antibodies produced by piglets induced by M13-DC3pep-GP5-B and M13-SCRMpep-GP5-B were not sufficient to produce protective effects.However,the level of neutralizing antibodies is low,and the serum exhibits neutralizing activity against PRRSV only at low dilution.To find and validate the new neutralizing epitope at the carboxy terminal of protective antigen GP5 protein from porcine reproductive and respiratory syndrome virus?PRRSV?,in this study the antigenicity of the carboxy terminus of PRRSV GP5protein was predicted by bioinformatics software,the coding sequence of amino acid168-198 at the carboxy terminus of GP5 protein was synthesized,then the recombinant phage M13-GP5 168-198 was constructed.The immunoreactivity of the GP5 168-198 polypeptide displayed on the surface of the recombinant phage was analyzed by western blot using clinically positive serum.The immunized antigen,prepared by 1.0x10¹³ pfu/mL recombinant phage mixed with aluminum hydroxide adjuvant was injected intramuscularly into the PRRSV antibody negative weaned piglets according to inactivated vaccine immunization program,,serum was separated for neutralization test with the clinical isolated virulent strain of PRRSV;The results of western blot showed that the GP5 168-198 aa polypeptide displayed on the surface of the recombinant phage had immunoreactivity,and the neutralizing antibody induced by the immunized piglets reached the highest in the sixth week,and the neutralizing titer was reached at 1:10.08.In summary,synthetic neutralizing B cell epitopes of PRRSV GP5 protein can be used to detect PRRSV antibodies in clinical pig serum;The neutralizing B cell epitopes was display by phage display technology,By immunizing weaned piglets,the level of neutralizing antibodies is low,and the serum exhibits neutralizing activity against PRRSV only at low dilution;The epitope of the carboxy terminal of GP5 protein predicted by biological software was display by phage display technology,By immunizing weaned pigs,the piglets could produce neutralizing antibodies,piglet serum showed good neutralization at the cellular level,which laid the foundation for the development of a novel multi-epitope vaccine of PRRSV GP5.