Prokaryotic Expression And Functional Research Of Ligand-binding Epitope Peptides Of VP2 Protein On IBDV

Infectious bursal disease (IBD), which usually occurs in 3～12weeks old chicken and young chicken, is a highly contagious and severe immunosuppression disease caused by infectious bursal disease virus (IBDV). The virus destroys the bursa of Fabricius, leading to serious damage to other immune organs and cells as well as considerable economic losses in poultry industries. IBDV can encode five kinds of proteins, which are VP1, VP2, VP3, VP4 and VP5 respectively. VP2 is the main structural protein. It is on the outside of nucleocapsid and carries the main neutrality epitope, which can induce host to generate neutrality antibody that will protect chicken from infecting by IBDV and owns ligand-combining epitope. The antibody plays an important role for virulence and cell tropism. The way for virus to infect cells is virus attachment protein (VAP) binds with cell receptors, which is virus receptors binding epitope of ligand or virus ligand binding epitope of receptor. If the target site of anti-virus drug can be found, the virus will be stopped before entering into cells or attaching its receptor, therefore virus are unable to enter cells. It plays a key role to prevent IBD and provide theoretical basis to further understand molecule mechanism of IBDV.ligand-combining epitope of VP2 protein was screened and identified before in my lab, and P22 polypeptides, which was ligand-combining epitope polypeptides, was obtained. In order to deeply research on P22, gene sequences of P22 (43AA)were synthesized based on the former study and E.coli codon optimize. Then the two gene sequences were linked together and induced to express in E.coli after cloning, enzyme digestion and linkage. The polypeptides could combine CEF. Its ability would increase when there were more polypeptide.To study the biological function of ligand-combining epitope peptide of VP2 protein and the receptor of IBDV, two polypeptides named P22 and P221, which were main protective antigen on VP2 protein, were synthesized based on amino acid sequence of IBDV and Fmoc solid phase synthesize principle based on three-dimensional structure of IBDV VP2 protein and right result of prokaryotic expression. The results were examined by mass spectrograph (MS), polypeptides binding CEF cells, and immunohistochemical assay and showed P22 and P221 could bind specifically CEF as ligand site of IBDV and its ability would increase when there were more polypeptide. Polypeptide immunology, anti-polypeptide serum binding IBDV and anti-polypeptide serum blocking IBDV were taken using CEF as target cell. The polypeptides P22 and P21 were conjugated using SMCC method based on the principle of ligand and receptor. Polyclonal antibodies were obtained and identified with ELISA and immunohistochemical assay. Results showed that the titers reached to 1:105, and it could also interact with IBDV-positive serum, the titer was 1:104. The anti-P22 polyclonal antibody serum could effectively bind the P22 peptide, and the capability was increased when the amount of polyclonal antibody serum was added.P22 polypeptide of ligand-combining epitope peptide of IBDV was obtained successfully in this study and could bind CEF effectively accord to experiment. Polyvalent antiserum of P22/P221 which was highly specifically was gained through polypeptide immunology. The antiserum could effectively bind the P22 peptide and could also neutralize the ability for IBDV to binding CEF. The results showed the polypeptide is the ligand-combining epitope of VP2 protein of IBDV and could suppress the infection of IBDV to CEF, which played a key role to study theoretical basis to further understand molecule mechanism of IBDV and provided foundation for designing the drug target.