Chen Gang, Yin You-Ping, Yuan Qing, Xia Yu-Xian, Wang Zhong-Kang
Key Laboratory of Gene Function and Regulation at Chongqing, College of Bioengineering at Chongqing University, Chougqing 400030, China.
Wei Sheng Wu Xue Bao. 2007 Dec;47(6):1066-9.
Citrus bacterial canker disease caused by the gram negative bacterium Xanthomonas axonopodis pv. citri (XAC) is a severe bacterial disease of most commercial citrus species and cultivars around the world. Single chain variable fragment (ScFv) is artificial construction small molecular antibody produced by genetic engineering which may be used to identify target pathogens and prevent plant diseases including XAC. To express ScFv against XAC and obtain functional ScFv, single-chain antibody fragments (ScFv 95) selected by ribosome display was amplified using an assembly of polymerase chain reaction (PCR), and a recombined plasmid pET30a( + )-XAC-ScFv was constructed by inserting the single chain Fv gene into bacterial expression vector pET30a( + ). PET30a( + )-XAC-ScFv was transformed into Escherichia coli BL21 (DE3) and expressed which was induced by IPTG. Products were purified though Ni-NTA His Bind resin. The collected antibodies were refolded by gel filtration chromatography, and activity assaying process was done. The results showed that ScFv recombined antibody of XAC with a molecular of 32kDa was expressed successfully as inclusion bodies and the functional ScFv was obtained through purification and renaturation. Meanwhile, the Biacore analysis indicates that XAC-ScFv-95 showed significant affinity to LPS of Xac, which paves a new way for immunization diagnosis and exploration of integrated control of citrus bacterial canker disease.
由革兰氏阴性细菌柑橘溃疡病菌（Xanthomonas axonopodis pv. citri，XAC）引起的柑橘溃疡病是一种严重危害全球大多数商业柑橘品种的细菌性病害。单链可变片段（ScFv）是通过基因工程生产的人工构建小分子抗体，可用于识别目标病原体并预防包括XAC在内的植物病害。为了表达抗XAC的ScFv并获得功能性ScFv，利用聚合酶链反应（PCR）组装扩增通过核糖体展示筛选出的单链抗体片段（ScFv 95），并将单链Fv基因插入细菌表达载体pET30a(+)中构建重组质粒pET30a(+)-XAC-ScFv。将pET30a(+)-XAC-ScFv转化到大肠杆菌BL21(DE3)中并经IPTG诱导表达。产物通过Ni-NTA His Bind树脂进行纯化。收集的抗体经凝胶过滤层析复性，并进行活性测定。结果表明，成功表达了分子量为32kDa的XAC重组ScFv抗体包涵体，经纯化和复性获得了功能性ScFv。同时，Biacore分析表明XAC-ScFv-95对Xac的脂多糖具有显著亲和力，为柑橘溃疡病的免疫诊断和综合防治探索开辟了新途径。
