Objective        To investigate the nanobodies specifically against the extracellular domain of prostate-specific membrane antigen (PSMA).       Methods        The extracellular domain of PSMA was recombined in eukaryotic HEK-293 cells through RT-PCR. With the recombinant protein as coating antigen

the solid phage selection was used in biopanning of nanobody phage display library. And the phage-ELISA was employed to screen out the phage clones with binding activity. Finally

cell ELISA and immunofluorescence technology were applied to verify the above phage clones.       Results        Sequencing indicated that the recombinant DNA sequence coding extracellular domain of PSMA was correct

and Western blot assay proved the successful expression of the protein. The phage clones with binding activity had got effective enrichment by 4 cycles of biopanning

and the positive ratio was increased from 8.3% to 64.6%. Cell ELISA showed that the absorption of PSMA-positive cells was significantly higher than that of PSMA-negative cells (0.621±0.043

vs

0.148±0.014

P

<

0.01). Immunofluorescence assay also showed that the phage clone from the above bound specifically to the PSMA-positive LNCaP cells.       Conclusion        Our phage clones derived successfully from the natural nanobody phage library are specially against the extracellular domain of PSMA in molecular and cellular levels.