Establishment And Application Of An ELISA For Detection Of IgM Antibody Against Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS)is an acute and highly contagious viral disease caused by porcine reproductive and respiratory syndrome virus(PRRSV)infection,which is characterized by symptoms in the reproductive system of sows and the respiratory system of piglets.PRRS is one of the most important infectious diseases faced by the global pig industry.It has been listed as a priority disease for elimination in the mid-and long-term animal disease prevention and control program of China.Enzymelinked immunosorbent assay(ELISA)is a widely used antibody detection method.Ig M is the first antibody produced in the body’s antiviral immune response.Detection of Ig M by ELISA can achieve the purpose of early diagnosis.However,there is no diagnostic method for PRRS Ig M.Therefore,there is an urgent need to establish a rapid,sensitive,suitable for the detection of a large number of samples and early infection detection method to assist the prevention,control and elimination of PRRS.The N and GP5 proteins are the main immunogenic proteins of PRRSV,which can induce Ig M production and are widely used to develop ELISA kits.Therefore,the aim of this study was to establish an indirect ELISA for PRRSV Ig M.To this end,the project carried out the following work:1.Expression and purification of recombinant PRRSV N protein and GP5 proteinAccording to the gene sequence of the highly pathogenic PRRSV strain H-1 isolated in our laboratory,the antigenic epitopes of N protein were analyzed.The genes coding amino acid residues 23-92(aa)and 112-122(aa)of N protein were connected by GGGGS linker sequence and expressed by Nm.The gene coding amino acid sequences 27-52 aa,119-127 aa,151-159 aa and 170-200 aa of GP5 protein were concatenated through GGGGS linker sequence and named GP5 m.After codon amino acid optimization,the corresponding gene was directly synthesized and ligated into the p GEX-6p-1 vector.After transformation and sequencing identification,p GEX-6p-1-Nm and p GEX-6p-1-GP5 m colonies were selected to express Nm and GP5 m proteins,namely Nm and GP5 m prokaryotic expression systems.The results of SDS-PAGE and Western blot showed that the p GEX-6p-1-Nm expression system could express and purify Nm protein with a concentration of 0.53 mg/m L.Use online website(https://www.novopro.cn/tools/)analysis of the highly pathogenic PRRSV strains JXA1,classic PRRSV strains DQ355796,NADC30 strain JN654459 N protein and GP5 protein amino acid sequence,It was found that the amino acid sequence7-29 aa,60-82 aa and 106-128 aa of GP5 protein contained transmembrane regions,and 1-31 aa contained signal peptides.The transmembrane region and signal peptide of GP5 protein were truncated,and the remaining sequence was concatenated through GGGGS linker sequence and named GP5 s.The corresponding gene was directly synthesized with the full-length N protein after aa codon optimization and connected to the p Fast Bac1-HTA vector.After transformation and identification,p Fast Bac1-HTA-N and p Fast Bac1-HTAGP5 s,namely insect cell expression systems of N and GP5 s.SDS-PAGE and Western blot results showed that the concentration of purified GP5 s protein was 0.65 mg/m L,and it could specifically react with PRRSV-positive serum.2.Establishment and preliminary application of ELISA methodUsing Nm protein and GP5 s protein as coating antigens,the indirect ELISA for PRRSV Ig M was established by optimizing the coating concentration of antigen,blocking solution and blocking time,dilution of test serum and incubation time,dilution of secondary antibody and incubation time,and color development time.Nm Ig M-ELISA and GP5 s Ig MELISA,respectively.The optimal reaction conditions for Nm Ig M-ELISA were as follows:0.1 μg/well Nm protein,coated at 4℃ overnight;The cells were blocked with 5% BSA at37℃ for 2 hours.The test serum was diluted 160 times and incubated at 37 ℃ for 1.5h.The secondary antibody was HRP labeled sheep anti-pig Ig M,diluted at a volume ratio of1:12000,and incubated at 37℃ for 1 hour.Finally,TMB was used for color development at room temperature for 15 min.The optimal reaction conditions for GP5 s Ig M-ELISA were as follows: 0.05 μg/well GP5 s protein,coated at 4℃ overnight;The cells were blocked with 5% BSA at 37℃ for 2 hours.The test serum was diluted 160 times and incubated at37 ℃ for 1h.The secondary antibody was HRP labeled sheep anti-pig Ig M,diluted 1:10000volume ratio,and incubated at 37℃ for 1 hour.Finally,TMB was used for color development at room temperature for 10 min.The results showed that the positive cut-off value of Nm Ig M-ELISA was 0.38,and the positive cut-off value of GP5 s Ig M-ELISA was0.49.The reproducibility of Nm Ig M-ELISA and GP5 s Ig M-ELISA was good,and the two ELISA methods did not cross react with the positive sera of PEDV,PRV,PCV,ASFV and other pathogens,indicating that the specificity of the two ELISA methods was good.Antibodies to NADC30 strain were detected in 198 positive pig sera on days 0,3,7,10,14,21,28 and 45 of infection.The positive rates of Nm Ig M-ELISA,GP5 s Ig M-ELISA and Ig G commercial kits were 0%,66%,92%,94%,96%,90%,80% and 64%,respectively.0%,40%,76%,80%,66%,60%,68% and 66%;0%,0%,5%,37%,95%,100%,95%,and100%.These results indicate that the Nm Ig M-ELISA established in this study is more suitable for PRRSV Ig M detection than the GP5 s Ig M-ELISA.Analysis of antibody detection levels in different infection stages showed that Ig M antibody could be detected by Nm Ig M-ELISA at 7 days after infection,which was earlier than Ig G antibody detected by commercial kits.A total of 182 clinical sera were tested with Nm Ig M-ELISA and Ig GELISA commercial kits.The results showed that Nm Ig M-ELISA had a good clinical detection effect.In addition,Nm Ig M-ELISA can detect PRRSV specific antibodies in the early stage of infection compared with Ig G-ELISA,which is a promising method for early diagnosis of PRRSV infection.