Epitope Identification Of Porcine Reproductive And Respiratory Syndrome Virus By Phage Display

Epitope, or antigenic determinant, is an antigen domain with affinity to antibody in Ag-Ab complex. Epitopes play a very important role in the structure and function of protein antigens. Precise epitope mapping may help design more reasonable vaccines and diagnostic reagents. A multi-epitope vaccine can provide protection against mutable virus because it may carry a broad spectrum of neutralizing epitopes. In addition, epitope-based vaccines can be easily distinguished from wild-type viruses, which facilitate the eradication of virial diseases. Phage-displayed peptide libraries technology provides a new approach to disclose protein structure and function, and has been used to map large number of epitopes.Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease of pigs characterized as severe respiratory disorders in piglets and widespread abortions in gestating sows. It was first reported in the United States in 1987. PRRS is epidemic in most swine-producing countries, and today it is associated with major economic losses. In our country, PRRSV CH-la strain was first isolated in 1996, but no information regarding epitopes is available so far. To explore the structure and function of viral proteins, we mapped epitopes in the N protein of PRRSV CH-la strain by phage display.Three hepta-peptides, STTPFKK, TKHPQFV and STGNRLT, were screened from a random peptide library by biopanning using a monoclonal antibody (MAb N3H2) against PRRSV N protein. These peptides are rather different from each other and show no homology with CH-la N protein. So, they represent 3 mimotopes.To identify native epitope of N protein, we constructed the ORF7 gene-targeted peptide library and screened by the MAb N3H2. After 3 rounds of biopanning, 3 clones were obtained. They share a consensus sequence IQTAFNQGA, which corresponds to amino acid (aa) 79-87 of CH-la N protein. A fragment coding IQTAFNQGA was synthesized and expressed, and the expressed GST-fusion protein reacted to the MAb N3H2 in Western blot and indirect ELISA. We conclude that this nonapeptide is a linear epitope, named Ep703.The sequence of the epitope Ep703 is identical to aa 80-88 of LV strain N protein, which is component of a conformational epitope consisting of aa 51-67 and aa 80-90 (Meulenberg et al. 1998). There were 2 amino acid mutations in aa 50-66 of CH-la N protein compared to aa 51-67 of LV strainN protein. In addition, we also noticed that the position of disulfide bond formation was different between the two strains. So we confer that the difference of antigenicity lies in the difference of the triple structure of proteins. The secondary structure of Ep703 is composed of an alpha-helix and a beta-sheet. The 3D structure of N protein shows that the domain of Ep703 is exposed on the surface of the protein, making the epitope to be readily recognized by antibody. No homology is found between the mimotopes and native epitope. However, they all contain high content of hydrophile amino acids and low content of hydrophobile amino acids.In summary, we obtained three mimotopes recognized by a MAb against PRRSV N protein, and mapped a linear B cell epitope located at aa 79-87 of N protein. This linear epitope is highly conserved among North American strains.