| Interferons (IFNs) are important cytokines which are used clinically as antiviral and antitumor agents. Binding of IFN to a cell surface receptor mediates activation of the signal transduction pathway that elicits the biological functions by inducing the transcription and expression of IFN-related genes. However, the recognition and interaction of proteins with others are finished through the interaction among several amino acids in the peptide fragments. Many researches demonstrate that short peptide ligands can mimicry the function of the corresponding whole protein molecules. In general, whole proteins are not suitable drug candidates for a number of reasons, including susceptibility to proteolytic degeneration, antigenicity and high cost. Beside those shortcomings, efficient treatment by IFNs is also hampered by various side effects. Since the peptide agonist or antagonist of cytokines has the advantages including low molecular weight, easy to synthesize, without antigenicity as well as reduced side effects, it has attract great attention of both researchers and investors. In this paper, using phage random peptide library technology, the polyclonal anti-IFN-α2b antibodies and WISH cells naturally carrying IFN receptors on the cell membranes were chosen as targets to study the antigen epitope of IFN-α2b and isolate the mimetic or antagonist peptide of IFN-α2b, which is significant in researching on the functional mechanism of IFN-α2b as well as the minimolecular mimic design and development of IFN-α2b.The polyclonal anti-IFN-α2b antibodies binding to immunomagnetic microspheres were chosen as targets and the biopanning of a 12-mer phage random peptide library was carried out. After 4 rounds of effective screening, the positive clones were characterized by ELISA and the positive rate was 53 % (49/92), which demonstrated that the positive clones specifically binding to antibodies were enriched. 12 clones randomly picked from the positive clones were sequenced. The homologous analysis of amino acid sequences of positive clones with IFN-α2b showed that 3 groups homologous to the 3 epitopes with strong antigenicity of IFN-α2b were obtained, defined by residues 1~13, 35~47 and 98~110 of IFN-α2b respectively. The results were anatomizing to the antigenicity predicting of IFN-α2b. Antibody blocking and phage immunity tests suggested peptides displayed on the positive phage could simulate, partly, the different epitopes of IFN-α2b in aspect of immune and antigen reactivity. Analysis on the three-dimensional structure of IFN-α2b showed that all 3 mimetic epitopes exposed to outsides of the whole molecular, which was easy to recognize by the antigen receptor of the antigen presenting cell. Especially the sequences of group II exhibited structural homology with Loop AB (29-35) of IFN-α2b involving binding to IFN receptor. It was supposed that neutralizing antibodies against AB loop produced after using IFN affected its biological function directly.Using gene recombinant technology, the prokaryotic expression vector pET32a(+)/GFP-IFN-α2b was successfully constructed and the fusion protein was expressed in E.coli BL21. SDS-PAGE and Western blot analysis showed that the fusion protein GFP-IFN-α2b retained the antigenicity of IFN-α2b. The receptor binding experiments indicated that the fusion protein with GFP activity could bind especially to WISH cells containing IFN receptors. The antiviral assay showed that the fusion protein possessed the IFN functions of antiviral activity and the antiviral activity was 1.0×107IU/mg. All the results demonstrated that the fusion protein was a bio-functional one which had not only the green fluorescence but also the IFN-α2b activity. As a simple and visualized material, the fusion protein we prepared here could be used to study the interaction of IFN-α2b with its receptor at the cell or molecular level. Meanwhile, the results laid the foundation for the research on the metabolism and function of IFN-α2b in methodology.WISH cells naturally carrying IFN receptors and polyclonal anti-IFN-α2b antibodies were chosen as targets and the biopanning of a 7-mer phage random peptide library was carried out by competitive elution method. The positive clones specifically binding to both WISH cells and antibodies were enriched and the positive rate of the last round was 51.1 % (23/45). 10 clones randomly picked from the positive clones were sequenced and the homologous analysis of amino acid sequences of positive clones with IFN-α2b was done. The corresponding amino acid sequences suggested 3 groups homologous to the 3 domains of IFN-α2b, defined by residues 24~41, 43~49, and 148~158 of IFN-α2b respectively. As represents corresponding to IFN receptor-binding domains, AB loop and E helix, clone No 26 and 35 were chosen for further characterization. Competitive ELISA and immunohistochemistry tests showed that clone No 26 and 35 could compete with IFN for binding to WISH cells and antibodies. Two peptides corresponding to clone No 26 and 35, designated SP-7(SLSPGLP) and FY-7(FSAPVRY) were synthesized. The receptor binding experiments showed that the two peptides could compete with GFP-IFN-α2b for binding to its receptor and the IC50 value was 8.90μg and 3.22μg respectively, which demonstrated that they could mimicry, partly, epitopes of IFN-α2b in charge of binding to receptor. The effects of two synthesized peptides to the antiviral activity induced by IFN were analyzed through CPE method. As a result, when the added amount was from 6.25μg to 100μg, both peptides could inhibit the IFN-induced antiviral activity in a dose-dependent manner. The IC50 values of both peptides were approximate 25μg, which suggested that they were antagonist peptides of IFN-α2b suppressing the antiviral activity mediated by IFN-α2b.In order to obtain the peptides mimicking IFN activity, on the basis of cell selection described above, additional 3 rounds of functional screening of the peptide library binding to WISH cells were proceed. The positive clones possessing the IFN functions of antiviral activity were enriched and the positive rate of the last round was 1.98 %( 20/1012). 10 clones randomly picked from the positive clones were sequenced and the homologous analysis of amino acid sequences of positive clones with IFN-α2b was done. The corresponding amino acid sequences suggested 4 groups homologous to the 4 domains of IFN-α2b, defined by residues 31~37, 68~74, 93~121 and 132~161 of IFN-α2b respectively. Of these positive clones, No.T9 (31~37) in group I and No.T3 (143~149) in group III overlapped, partly, with AB Loop (26~35) and E helix (141~158) of IFN-α2b respectively. Especially 2R and 7R of IR-7 were identical to 144R and 149R of IFN-α2b which were so called"hot-spot"residues very important for binding of IFN to its receptor. Competitive ELISA and immunohistochemistry tests showed that No T9 and T3 could compete with IFN for binding to its receptor. Two peptides corresponding to clone No T9 and T3, designated KP-7(KNVHPPP) and IR-7(IRPDTPR) were synthesized. The receptor binding experiments showed that the two peptides could compete with GFP-IFN-α2b for binding to its receptor and the IC50 value was 13.43μg and 9.49μg respectively, which demonstrated that they could mimicry, partly, epitopes of IFN-α2b in charge of binding to receptor. The antiviral assay showed that when the added amount was from 1.25μg to 5μg, both peptides could have the IFN-induced antiviral activity in a dose-dependent manner, which suggested that they were mimetic peptides of IFN-α2b. Furthermore, the antiviral activity of KP-7 was a little higher than IR-7 and there was a cooperation when both peptides were added together. It assumed that multi-sites interaction of IFN to its receptor caused to the IFN-induced antiviral activity.In summary, the valences and characteristics of IFN-α2b antigen epitopes had been understood comprehensively. It is helpful in laying the foundation for the molecular mechanism of the interaction of IFN with its receptor and the preparation of monoclonal antibody against specific epitopes. Furthermore, the screening systems such as whole cell together with antibodies or functional screening strategies had been developed, which presented a new method in studying the molecular mechanism of IFN function and screening the mimetic or antagonist peptides of cytokines as well as developing the minimolecular mimics of IFN-α2b.
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