Antigenic Epitope Mapping Of Classical Swine Fever Virus (CSFV) Immunogenic Protein E2 By Phage Display Peptides Library

Phage display describes a selection technique in which a peptide or protein is expressed as a fussion with a coat protein of bacteriophage, resulting in display of the fused protein on the surface of the virion, while the DNA encoding the fussion resides within the virion. Phage display has been used to creat a physical linkage between avast library of random peptide sequences to the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules (antibodies, enzymes, cell-surface receptors etc) by an in vitro selection process called panning.In this experiment, to screen the epitope of E2 protein of classical swine fever virus (CSFV) defined by the monoclonal antibody (McAb) All, which had been prepared in previous experiment, the McAb All was raised in mice and followed by purification, the concentration of protein was assayed by using the BCA protein assay kit. The random 12-mer peptide library with 2.7 X 109 complexity and 4.6 X1013 titering respectively was used in biopanning. At first, 1.67 u g per well McAb All was coated on three wells of a plate, and then 1.5 X 1011 phage virion was diluted and added, after incubating with the target, wash away unbound phage by TBST(0.1% Tween-20), the bound phage was eluted with pH 2.2 Tris-Gly buffer and amplified, the specially bound phage was enriched by taking through addition binding/amplification cycles.ln the following cycles, the stringency of panning can be increased by raising the concentration of TBST or decreasing that of McAb All, collecting and titering the washing phage of last time and output phage in each round, the selective ratio and the false positive rate of each round were worked out, the gradually increasing of selective ratio and decreasing of positive rate shows that the panning was effective. After 4 rounds of panning, 11 phage clones were selected after competitive-ELlSA, the DNA samples of 8 positive clones and 1 negative clone were sequenced and all the foreign peptides inserted was also deduced, a clear consensus binding sequence emerged. Compared with the amino acidequence of E2 protein, the consensus were homogeneous to the 28-35 amino acid of E2 protein. In order to verify this result, the Sandwich-ELISA and Western-blotting test were employed, all phage clones with core sequence are positive, while clone without core sequence is negtive. It indicates that the core sequence of the foreign peptides can be recognized by MAcb All, the core sequence is mimic epitope of E2-protein. A synthesised peptide which contain a partial sequence amino acid residues was used in I-ELISA, the result is also positive, it means that the amino acid residues 28-35 of E2 protein is likely a linear epitope recognized by McAb All.