中国生物工程杂志

Objective: To express PSMA (prostate-specific membrane antigen) specific multivalent nanobodies in the prokaryotic system, and preliminarily evaluate their biological properties. Methods: The multivalent nanobody expression vectors were constructed by the Bglbrick method, and then transferred into E.coli Rosetta(DE3) for protein production. SDS-PAGE and Western blot assays were performed to identify the purified nanobody samples. BCA (bicinchoninic acid assay) kits were used for expression yield determination. The specific binding affinities were evaluated by immunofluorescence and flow cytometry. The binding EC₅₀ values were detected by cell-based ELISA. The endocytosis efficiency was investigated via flow cytometry. Results: Recombinant E. coli strains for PSMA specific monovalent, bivalent, trivalent and tetravalent nanobodies production were successfully constructed, respectively. Fermentation results showed that all four kinds of nanobodies could be expressed as soluble proteins with high yields at shake flask level, among which the bivalent nanobody presented the highest expression yield [(259.14±23.56) mg/L], whereas the monovalent type lowest [(100.58±6.27) mg/L]. In the subsequent cell binding assays, results indicated that all four nanobody samples could specifically recognize and bind to PSMA-positive tumor cells. Notably, compared with monovalent nanobody, the binding affinity of bivalent, trivalent and tetravalent nanobodies were improved approximately 3.32-, 2.29- and 2.03-fold, respectively. Finally, the endocytosis experiments were conducted and the results suggested that all four kinds of nanobodies could be efficiently endocytosed by PSMA-positive tumor cells and the uptake rates in 0.5 h were all above 80%. Conclusion: PSMA specific multivalent nanobodies, especially PSMA specific bivalent nanobody, had higher expression yield and better binding affinity than monovalent nanobody. Meanwhile, they remained the same uptake level with monovalent nanobody. In this context, PSMA specific multivalent nanobodies could be a potential class of candidates for the development of PSMA-based therapies.