Zhang Qi, Bai Gang, Cheng Jiaqi, Yu Yangsheng, Tian Wang, Yang Wenbo
Department of Microbiology, College of Life Sciences, Nankai University, China.
Biosci Biotechnol Biochem. 2007 Jun;71(6):1514-20. doi: 10.1271/bbb.70022.
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.
由松材线虫（BX）引起的松树萎蔫病，也被称为松材线虫病（PWN），是松树最具毁灭性的病害。在本研究中，我们构建了一种针对松材线虫纤维素酶抗原（BXCa）的高度特异性抗体（单链可变片段，scFv）。该抗体是针对从能高效水解羧甲基纤维素的PWN中纯化得到的高抗原性纤维素酶产生的。从用BXCa免疫的BALB/c小鼠的新鲜脾脏中提取总RNA，经逆转录-聚合酶链反应后，用接头将V(H)和V(L)组装起来。最终的噬菌体展示抗体库库容约为5×10(4)。经过五轮亲和筛选，我们获得了针对BXCa的特异性工程抗体。阳性噬菌体克隆用于感染大肠杆菌HB2151，酶联免疫吸附测定和斑点印迹表明可溶性scFv能特异性结合BXCa。对scFv进行测序，并在与增强型绿色荧光蛋白融合的大肠杆菌BL21中表达，该融合蛋白具有绿色荧光和抗BXCa功能。利用该融合蛋白，我们通过倒置荧光显微镜和激光扫描共聚焦显微镜在活的PWN中定位纤维素酶。结果有力地表明纤维素酶是在食管腺细胞中合成的。这种在活的PWN中检测和定位蛋白质的新方法可能会加深我们对松树萎蔫病潜在病理学的理解。
