Generation Of Monoclonal Antibody Against Porcine Reproductive And Respiratory Syndrome Virus GP4 Protein And Its Antigenic Epitope Screening

Porcine reproductive and respiratory syndrome virus(PRRSV) was classified in the family of Arteriviridae in the newly created order Nidovirales. Typical clinical symptoms of PRRS were fever, abortion, prematurity, stillbirths and respiratory disease in pig blocks. GP4 protein, as one of the minor structural protein, is the constituent of the virion envelop encoded by the viral open reading frame 4(ORF4). GP4 is a heavily glycosylated protein of 180/178 amino acids which has 4 glycosylation sitesis, and exists at small amount in virion. GP4 could induce neutralizing antibodies, and has also significance in cell receptor recognition of virus particles, as well as virus mutation. The epitope research with phage display technology not only demonstrates the structure and function of antigen-specific molecule, but also reveals the mechanism of antigen-antibody reaction, and now has been widely used in the development of peptide vaccines and new drugs. In this experiment, with PRRSV HUN4 strain as the research object, it was undertaken to prepare the monoclonal antibody against PRRSV GP4 protein, screen and preliminarily characterize its antigenic epitopes.In this paper, PRRSV GP4 protein was prokaryoticly expressedand, and used as antigen to immunize Balb/C mice. With conventional cell fusion method, an anti-PRRSV GP4 protein monoclonal antibody 5F12 was successfully prepared after three rounds of subclone and screen. It was identified as Ig G2 a subclass, and had better stability and specificity, which not only responsed with recombinant PRRSV GP4 protein but also reacted with PRRSV.With phage display technique, phage random peptide library was panned with 5F12 monoclonal antibody for 4 rounds, and 10 affinity phage clones were harvested. Sequencing results showed that 4 consensus sequences of the 12 peptides were identified as AKFEVCSPVVLG, GVNQENMLHFSF, NPRIRLNFIRIG, and SGVYKVAYDWQH, and named as phage- â… - â…£ respectively. It was found the 12 peptide of phage-â… and â…¡ had high similarity with PRRSV(Hu N4) GP4 amino acid sequence of 112-123, and 84-95, respectively. Phage-based ELISA and antigen competitive inhibition test confirmed that the affinity phages displayed good reactivity with PRRSV and its GP4 protein. In general, this would facilitate the PRRSV diagnosis and development of epitope vaccines and provide solid theoretic and experimental basis for the study of biological characterization of PRRSV and comprehensive control of PRRS.