Preparation of anti-hCG antibody-like molecule by using a RAD peptide display system / 生物工程学报

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.

Screening the peptide high-binding VEGF receptor 3 with phage-displayed random peptide library / 第三军医大学学报

Objective To get a high affinity peptide of vascular endothelial growth factor receptor 3 (VEGFR3) as a potent carrier targeted to lymphangiogenesis of ovarian cancer via the technology of phage display. Methods Solid-phase was panned with direct VEGFR3 extracellular protein coating, then the unbound phage was washed away and the eluted phage was amplified. The positive phage clones were identified by ELISA and sequenced, and the affinity and specialty were identified by competent ELISA. Results After four-round bio-panning, the enriched positive phage clones were identified by ELISA. Eight positive phage clones were sequenced and 5 were consensus (WHGSLKQNLWWY). The short peptide displayed on screened positive phage could bind specifically to VEGFR3, and the binding could be inhibited by natural antibody VEGF-D. Conclusion The phage clone (phage-WHGSLKQNLWWY) obtained via bio-panning of peptide library has a high affinity with VEGF receptor 3. The peptide could be a potent carrier targeted to VEGFR3.