Use of an enhanced green fluorescence protein linked to a single chain fragment variable antibody to localize Bursaphelenchus xylophilus cellulase.

The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as pine wood nematode (PWN), is the most devastating disease of pine trees. In this study, we engineered a highly specific antibody (single-chain fragment variable, scFv) against B. xylophilus cellulase antigen (BXCa). The antibody was raised against highly antigenic cellulase purified from PWN that efficiently hydrolyzed carboxymethyl cellulose. Total RNA was extracted from fresh spleens from BALB/c mice immunized with BXCa, and V(H) and V(L) were assembled with a linker following reverse transcriptase-polymerase chain reaction. The final phage display antibody library had a repertoire of about 5 x 10(4). We obtained specific engineered antibodies against BXCa after five rounds of affinity selection. The positive phage clones were used to infect Escherichia coli HB2151, and enzyme-linked immunosorbent assay and dot blotting showed that the soluble scFv specifically binded to BXCa. The scFv was sequenced and expressed in E. coli BL21 fused to enhanced green fluorescence protein, which had both green fluorescence and anti-BXCa functions. Using the fusion protein, we located cellulase in live PWN using an inverted fluorescence microscope and a laser scanning confocal microscope. The results strongly suggested that the cellulase was synthesized in the esophageal gland cells. This novel method of detecting and localizing proteins in live PWN might further our understanding of the underlying pathology of pine wilt disease.