| Porcine reproductive and respiratory syndrome(PRRS)is an acute,highly contagious infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).The disease has been discovered for more than 30 years and has not been widely and effectively controlled so far.It is still one of the important pathogenic factors endangering the development of pig industry.Vaccine immunization is still a common method to control PRRS,so how to accurately evaluate the immune status of pigs is particularly important.The monitoring of neutralizing antibody is the key to evaluating the immune status of pigs.At present,the antibody level of N protein in pigs is mainly monitored at home and abroad to evaluate PRRS infection pressure and stable state of pigs.N protein antibody is not neutralizing antibody and cannot reflect the immune protection status of pigs.Therefore,neutralization test was used to measure the serum of pigs with different immune backgrounds in this study,which is helpful to understand the immune and infection status of pigs.In addition,among the structural proteins of PRRSV,GP2,GP3,GP4 and GP5 can induce the body to produce specific neutralizing antibodies,and the research on protective antigen of PRRSV mainly focuses on GP5 protein,while the research on GP2,GP3 and GP4 is less.Therefore,the consistency between serum antibody and neutralizing antibody of PRRSV secondary structural protein was also investigated in this paper.Experiment 1 was designed to evaluate the immune protection levels of pigs with different immune backgrounds to explain why PRRS remained stable in pig farms with low resistance stability.This study first used the lab N antibody detection kit for 4 with large-scale pig farms background immune serum antibody detection,and according to the N protein antibody level and farm production grades 4 pig farms can be divided into two categories: low resistance stability pig farms(N protein antibody,stable production performance pig)and high stable farm(high and stable production performance N protein antibody).At the same time,serum neutralization test was performed on selected serum samples from 4 pig farms.The results showed that the pigs still had high neutralization antibody in the condition of low N protein antibody(late or longer period of PRRSV infection and immunization).These results showed that PRRS remained stable due to the existence of protective neutralizing antibodies in pigs.Experiment 2 was designed to express truncated GP2,GP3 and GP4 recombinant proteins with missing transmembrane regions and signal peptides by prokaryotic expression system.In this study,according to the ORF2,ORF3 and ORF4 gene sequences of PRRSV HY21 strain,the specific primers were designed to amplify the truncated gene fragments and connect them to the prokaryotic expression vector p ET-28a(+)after the signal peptides and transmembrane regions were predicted and deleted by online software.The recombinant plasmid was transfected into EScherichia coli BL21(DE3)and the recombinant proteins GP2(23.3k Da),GP3(27.6k Da)and GP4(17.8k Da)were successfully expressed by IPTG induction,which were consistent with the expected protein size.The high expression of recombinant protein was obtained by optimizing the concentration of enrichment bacteria and induction expression time.The inclusion body protein was purified by urea method and KCl staining and gel cutting method.The recombinant protein was detected by Western blot using high PRRSV positive serum as primary antibody.The results showed that the recombinant protein could be recognized by high PRRSV positive serum,indicating that the recombinant protein had good reactivity.Experiment 3 was designed to explore the correlation between serum antibody and neutralizing antibody of PRRSV minor protein.The recombinant proteins GP2,GP3 and GP4 obtained in Experiment 2 were used as antigen coated ELISA plates,and the related indirect ELSIA method was optimized to detect antibodies against GP2,GP3 and GP4 in serum with different neutralizing titers.In the process of optimizing the ELISA method,it was found that only GP3 recombinant protein had good reaction with serum,while GP2 and GP4 recombinant protein had poor reaction with serum,indicating that the removal of signal peptide and part of the transmembrane region may affect protein reactivity,or protein cannot be expressed correctly in the prokaryotic system.As a result,the conformation and activity of proteins are not completely close to natural proteins.Therefore,only the indirect ELISA method of GP3 recombinant protein was optimized.The results showed that most of the GP3 positive sera had neutralizing antibodies.Although the antibody level of GP3 protein is not consistent with the neutralizing antibody,its positive rate has a certain correlation with the neutralizing antibody. |
