Screening Mimotopes Of Actinobacillus Pleuropneumoniae With Phage Display 12-Mer Peptides Library

Porcine contagious pleuropneumonia (PCP) caused by Actinobacillus pleuropneumoniae (APP) is a severe, contagious pulmonary disease of pigs. Mimotopes of APP and its 3 major Apx (Actinobacillus pleuropneumoniae RTX toxin) were explored from the phage display 12-mer random peptides library, and the immunogenicities of the different epitopes were identified after the screening. The results are as following:1. Screening mimotopes of Actinobacillus pleuropneumoniaeThe specific sera IgG were obtained by immunizing the rabbit with Actinobacillus pleuropneumoniae vaccine, and purified through low-pressure chromatography system after rude purification. This IgG was used as target to screen the phage display 12-mer random peptides library for 3 rounds in different conditions. After 3 rounds of screening, the yield ratio increased from 5.76×10^-5 to 1.69×10^-2, and the P/N increased gradually. There were 6 phage clones identified by ELISA and they did not have cross-reactions with irrespective antibodies. Gradient ELISA of the positive phage clones showed a dose independent relationship. In order to wipe of the fake positive, the eluted phages were absorbed by the negative rabbit sera. DNA sequence assays showed that 5 of the 6 positive clones displayed the same peptide sequences.2. Screening mimotopes of 3 major Actinobacillus pleuropneumoniae RTX toxins The specific sera IgG of 3 major Apx(Apx I ,Apx II ,and ApxIII) were purified throughlow-pressure chromatography system after rude purification. These IgG were used as targets to screen the phage display 12-mer random peptides library in different conditions respectively. After 4 rounds of screening, the yield ratios and the P/N increased gradually.5 phage clones were identified by ELISA respectively and they did not have cross-reactions. During the selection progresses, the competitional elution implemented by Apx protein was used but not acid elution to guarantee the specifities of the positive clones. DNA sequence assays showed the high homologous between the phage epitopes and 3 major Apx.3. Identification of the immunogenicities of the positive phage clones Kunming mice were immunized by the different positive clones of phage, inactivatedAPP and Subunit bacteria vaccine. Specific antibodies of APP and Apx were detected by IHA and ELISA.