Screening And Identification Of Mimotopes Of Avian Infectious Bronchitis Virus

Infectious bronchitis virus (IBV) is causative pathogen of infectious bronchitis (IB), an acute, highly contagious respiratory disease in chickens. In this dissertation, two IBV mimotopes were screened from phage display peptide library. And the DNA sequences of mimotopes were inserted into the active site loop of thioredoxin which is itself inserted into flagellin gene, respectively. When induced, the fusion protein is expressed and displayed on the bacteria surface. Two recombinant bacteria have specific affinity to IBV antiserum. This study may provide a foundation for developing a new type vaccine and diagnostic tests for IB.Immunogtobulin G (IgG) of IBV was purified from chicken sera with caprylic acid and (NH₄)₂SO₄ The IgG was used as a target to screen the phage display random 12-peptide library. After 3 rounds of bio-panning, the enriched ratio of phage increased to 125 times. 50 phage clones were randomly picked and analyzed for the binding activity with ELISA. 22 positive clones which had specific affinity to IBV sera were obtained and identified by DNA sequencing and competitive inhibition ELISA. The results showed that the phage clones with peptide sequences KSPKHSSSALHF or SIIQMNLHRPTS could specifically inhibit the binding of IBV antigen with IBV antiserum. Compared with amino acid sequences of IBV in GenBank, there were no homologous with the two peptides sequences. The results indicated that the peptides might be mimotopes of IBV.The DNA fragments that coded the peptide sequences KSPKHSSSALHF and SIIQMNLHRPTS were cloned into the expression vector pFliTrx by means of genetic engineering technique, respectively. The recombinant plasmids p1 and p2 were constructed and transformed into an expression host E. coli GI826 respectively. And two recombinant E. coli F1 and F2 which displaying the IBV mimotopes on the bacterial flagellum were constructed successfully. The recombinant bacteria showed actual positive reactivity with IBV antiserum by antigen-antibody recognition in vitro. The results indicate that two IBV mimotopes still had specific affinity to corresponding IBV antibodies when they were departed from phages.It was concluded that the two IBV mimotopes may have a great potential application in developing new type diagnosis reagents and vaccines for IB.