Identification of Epitopes for Neutralizing Antibodies Against H10N8 Avian Influenza Virus Hemagglutinin / 中国医学科学院学报

Objective To develop neutralizing monoclonal antibodies (MAbs) against H10N8 avian influenza virus hemagglutinin and to identify the binding sites. Methods MAbs against hemagglutinin of H10N8 avian influenza virus were developed by genetic engineering. Neutralizing MAbs were screened by microneutralization assay,and then tested by enzyme-linked immunosorbent assay and Western blot to identity the binding sites.The homology modeling process was performed using Discovery Studio 3.5 software,while the binding epitopes were analyzed by BioEdit software. Results One MAb that could neutralize the H10N8 pseudovirus was obtained and characterized. Analysis about epitopes suggested that the antibody could bind to the HA1 region of hemagglutinin,while the epitopes on antigen were conserved in H10 subtypes.Conclusions One neutralizing antibody was obtained by this research.The MAb may potentially be further developed as a pre-clinical candidate to treat avian influenza H10N8 virus infection.

Eukaryotic expression and biological activity of human interleukin-35 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To express and purify recombinant human interleukin-35[IL-35-IgG1 (Fc) in eukaryotic expression system and to study the interaction of IL-35 with gp130 protein.</p><p><b>METHODS</b>A mammalian expression vector, pSTEP2-IL35-LFc, was constructed and transfected into HEK293T cells. Then rhIL-35-IgG1 (Fc) was expressed and purified with protein A affinity chromatography, and was examined with SDS-PAGE and Western blot analysis. The binding of IL-35 to its receptor gp130 was investigated using enzyme-linked immunosorbent assay (ELISA). The biological effect of IL-35 on gp130 was explored in M1 myeloid leukemia cells.</p><p><b>RESULTS</b>rhIL-35-Fc with high purity on reduced SDS-PAGE was obtained. ELISA confirmed that IL-35-Fc was bound to gp130 and neutralized the function of gp130 in M1 myeloid leukemic cells.</p><p><b>CONCLUSIONS</b>High-purity and biologically active rhIL-35-Fc protein successfully produced in this study. IL-35 binds to gp130 and neutralizes its activity of in M1 myeloid leukemic cells.</p>

Prokaryotic expression of recombinant human bone morphogenetic protein-2 and preparation of monoclonal antibodies / 中国医学科学院学报

<p><b>OBJECTIVE</b>To express and purify the recombinant human bone morphogenetic protein-2 mature peptide (rhBMP-2m) in prokaryotic system and to develop highly-specific monoclonal antibodies.</p><p><b>METHODS</b>An engineered E. coli strain expressing rhBMP-2m was fermented. The bacterial cells were firstly lysed and then the rhBMP-2m inclusion bodies were isolated by centrifugation. After the inclusion bodies had been solubilized by high-concentration denaturing agents, denatured rhBMP-2m was purified by cation ion-exchange chromatography. Biologically active rhBMP-2m was obtained by refolding of purified denatured rhBMP-2m through direct dilution. The refolded rhBMP-2m was used to immunize Balb/c mice to develop anti-rhBMP-2m monoclonal antibodies using classic hybridoma technique.</p><p><b>RESULTS</b>rhBMP-2m with a purity greater than 95% was obtained on reduced SDS-PAGE. The refolded rhBMP-2m was measured to be bioactive by the induction of alkaline phosphatase activity in MC3T3-E1 cells. Two hybridoma cell lines that stably secreted anti-rhBMP-2m antibody were developed from the immunized mice.</p><p><b>CONCLUSION</b>Bioactive rhBMP-2m protein and its monoclonal antibodies were successfully prepared, which will provides a solid base for future studies on rhBMP-2.</p>