抗松材线虫纤维素酶单链抗体库的构建及筛选

构建鼠源性松材线虫纤维素酶（Bursaphelenchus xylophilus cellulase, BXC）的噬菌体单链抗体库，从中筛选特异性BXC的单链抗体。以BXC为抗原免疫BALB/C小鼠，从脾脏提取总RNA，用RT-PCR技术扩增小鼠抗体重链（V_H）和轻链(V_L)可变区基因。经重叠PCR(SOE-PCR)在体外将V_H和V_L连接成单链抗体(scFv)基因，并克隆到噬菌粒载体pCANTAB5E中，电转化至大肠杆菌TG1，经辅助噬菌体超感染，成功构建了库容为5×10⁴的Anti-BXC单链抗体库，并从该抗体库中初步筛选到了特异性识别BXC的噬菌体单链抗体scFv。将表面展示单链抗体的单克隆噬菌体转化大肠杆菌HB2151进行可溶性表达，SDS-PAGE及Western blot分析结果显示，可溶性scFv获得表达，且与BXC具有结合活性，为松材线虫的检验检疫以及病理学研究奠定了基础。

Construction and Screening of Phage Display Single Chain Antibody Library Against Bursaphelenchus xylophilus Cellulase

A phage display single-chain variable fragment (scFv) library against Bursaphelenchus xylophilus cellulase (BXC) was constructed and used to screen the specific antibodies binding to BXC. The total RNA was extracted from fresh spleens of BALB/C mice immunized with BXC. Gene fragments encoding VH and VL were amplified by RT-PCR and assembled into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The recombinant fragments were cloned into the phagemids (pCANTABSE) and electroporated into E. coli TG1. The recombinant phagemids were rescued by reinfection of helper phage M13K07. The repertoire of the phage display antibody was about 5 x 10(4). The specific antibodies against BXC were obtained after five rounds of affinity selection. The positive phage clone was used to infect E. coli HB2151. SDS-PAGE and western blot analysis showed that the soluble scFv antibodies expressed bound specifically to BXC. The studies laid foundation for quarantine and pathological study of Bursaphelenchus xylophilu.