Preparation Of A Monoclonal Antibody With Neutralizing Activity Against North American PRRSV GP4 And Identification Of Its Epitope

Porcine reproductive and respiratory syndrome virus is the pathogen of PRRS.NADC30-like strains belong to subgroup 5 of PRRSV have been prevalent in China since 2013.Glycoprotein GP4 of PRRSV plays an important role in infecting PRRSV and inducing neutralizing antibody,but there are very few reports on the preparation and research of GP4MAb with neutralizing activity.The purpose of this study was to prepare a monoclonal antibody（MAb）against PRRSV GP4 protein with neutralizing activity and to identify the antigenic epitopes recognized by MAb.This was done to understand the structure and function of PRRSV genome,and it is significant in the further use of neutralizing monoclonal antibody for immune prevention and treatment of PRRS.In this study,the purified PRRSV SD53 strain was used to immunize6-week-old BALB/c female mice three times.Seven days after the third immunization,the positive result from IFA of the serum of the mice showed that they were immunized.The spleen cells of the positively immunized mice and the myeloma SP2/0 cells were fused together.After four times of sub cloning,the stability of the antibody secreted by the selected hybridoma cells were verified.The results of IFA showed that a positive hybridoma cell line that can stably secrete monoclonal antibody（MAb）against PRRSV was obtained and was named LM26.A large number of positive hybridoma cells were cultured and ascites were prepared.The monoclonal antibodies in ascites were collected and purified by affinity chromatography.Western blot results showed that purified MAb were obtained.The titers of cell culture supernatant and ascites detected by IFA were 1:16 and 1:1600,respectively.The MAb had neutralizing activity against North American PRRSV SD53 strain,the lowest neutralization titer was 1:6 and the highest was 1:50.The antibody subclass was identified as IgG2a,and light chain wasκchain.This study further defines the LM26 as a PRRSV GP4 monoclonal antibody and accurately identified its recognized epitopes.Firstly,the nucleic acid sequence of a series of structural proteins of PRRSV SD53strain were amplified and then recycled,and cut by two endonucleases,and then digested into pGEX-6P-1 expression vector.A series of recombinant expression plasmids were constructed,and used to induce and express the recombinant protein.A series of fusion proteins of structural protein peptides with GST were obtained.Western blot results showed that a series of recombinant proteins were successfully expressed,and LM26 only specifically recognized recombinant the GP4 protein.Secondly,the recombinant plasmid pCAGGS-FLAG-GP4 after correct sequencing was transiently transfected into 293T cells as detection antigen,and the expression of recombinant GP4 protein was verified to have been expressed successfully by IFA which reacted with LM26,and proved that the monoclonal antibody against PRRSV GP4 had been successfully prepared.Finally,the epitope of the monoclonal antibody against GP4 protein was identified,and western blot results showed that LM26 can recognize aa 57^aa 62(⁵⁷VVLQDI⁶²)of the GP4 protein.The sequence ⁵⁷VVLQDI⁶² was compared with amino acid sequence of GP4from other 28 PRRSV strains data downloaded from GenBank from domestic and abroad.The results showed that GP4 protein aa 57^aa 62was highly conserved in HP-PRRSV strains and NADC30-like PRRSV strains,and there were two mutation sites in classical PRRSV strains,which were V⁵⁷A and Q⁶⁰H,respectively;there was a mutation site V⁵⁷M in European PRRSV strains as well.However,results from IFA showed that LM26 reacted with vaccinated strains HuN4-F112,CH-1R,MLV and TJM-F92,but not with European strains DV and VP046 BIS,suggesting that V⁵⁷A and Q⁶⁰H mutations did not affect the epitopes recognised by LM26.This epitope is relatively conserved in North American PRRSV.Overall,in this study,a monoclonal antibody with neutralizing activity against PRRSV GP4 protein was successfully prepared and it identified the epitope located in GP4 protein as ⁵⁷VVLQDI⁶².This research is helpful in the further study of the structure and function of PRRSV genome.