Screening And Characterization Of Infectious Bronchitis Virus S1 High-affinity Peptide And Its Related Receptor Function

Avian infectious bronchitis is an acute, highly contact respiratory infectious diseases caused by avian infectious bronchitis virus. The serotype of IBV is complicated for it could mutate easily, so the clinic features of this diseases have great differences. Therefore, the studies of IBV molecular biology and its receptor of invasion are important. Spike protein of IBV is a structure protein locates at the surface of virus particle, it has epitopes to induce host protective antibody, and receptor binding domain to mediate virus invasion. It is a key protein of coronavirus invasion and production.1. This study constructed recombinant prokaryotic expression plasmid pET-S1 with S1 gene from IBV Beaudette, and expressed in E. Coli Rosetta, western bolt with IBV polyclonal antibody proved that target bind of 63 Ku is IBV S1 protein.2. Using IBV S1 protein as target protein, we carried out four rounds of biopanning with phage displayed 12-mer peptide library.10 phages with high affinity with S1 protein were gotten and their nucleotide sequences were measured, the related polypeptide was synthesized. The result revealed that there were three phages could bind with S1 specifically:LWAQAKSLHTLA, MYSPRPPALSTV, HWDPFSLSAYFP, we named them polypeptide L, M, H, respectively. ELISA proved that polypeptide H could competitively binded with S1 against gAPN; it also binded with polyclonal antibody of gAPN, which proved that polypeptide H had simula epitopes of gAPN. Virus inhibition test in vitro proved that polypeptide H could inhibit virus from infecting Vero cells in 59.6% with concentration of 500 μg/mL.3. We established an efficient high-affinity phage-mediated ELISA to detect IBV, the minimum virus amount could be detected is 1.21 ×105 copeis/μL. We compared this ELISA with RT-PCR and Real-Time PCR, the result revealed that Real-Time PCR was the most sensitive one and ELISA was the least.4. Immunofluorescence of FITC-labelled polypeptide H proved that the polypeptide H could effectively bind with IBV. With this labelled polypeptide, the distribution of virus in different tissues of chicken infected with IBV M41 strain was assayed. The results revealed that virus could be detected in trachea, lung and kidney at 1,3,3 days post infection (PI) respectively, and IBV could be detected in trachea at 17 days PI, and 7 days PI in lung and 10 days PI in kidney. The same assay with rabbit anti-IBV polyclonal antibody showed that positive polypeptide is more sensitive.5. We cloned aminopeptidase N (gAPN) with 2906 bp in length, constructed recombinant prokaryotic expression plasmid pET-gAPN, and expressed in E. Coli Rosetta. We prepared polyclonal antibody with purified gAPN recombinant protein, western-blot proved that it had good biological activities to react against prokaryotic expressed and natural gAPN; western-blot also proved that prokaryotic expressed gAPN can bind with IBV. Co-immunoprecipitation test proved that gAPN antibody could precipitate S1 protein which lied foundation to prove gAPN was functional receptor of IBV. Polyclonal antibody against gAPN can inhibit virus from infecting CEK calls at the 21 dilution, the inhibit capabilities is 81.14%.6. The eukaryotic expression plasmid pcDNA-gAPN was constructed and transferred into Hela cells. The results of indirect immunofluorescence, RT-PCR, and Real-Time PCR indicated IBV Beaudette strain could infect Hela cells transfected with gAPN gene and produced infectious virus particles. With immunofluorescence technique and laser confocal microscopy observation, it proved that gAPN can mediate IBV infect host cell, the fluorescence of gAPN located in cytomembrane, the fluorescence of virus were in cytoplasm. Different strains of IBV, including HI20, W93, M41, HH06 and HH12, were used to infect Hela cells tranfected with gAPN gene and all got positive results. To recongnize the epitope in gAPN, We constructed different eukaryotic expression plasmid of pCDNA-gAPN945 and pCDNA-gAPN 1695 to express gAPN in short segments, the results showed that it should locate between 945-1695bp in gAPN. After compared the 3-dimensioned structure of pAPN, we confirmed 3 sites locates inβcorners are possible to be binding sites of IBV S1. This lied foundation of final predication about binding sites of gAPN.