柑桔溃疡病菌重组单链抗体的高效表达及活性检测

High efficient expression and bio-activity assay of recombinant antibody for citrus bacterial canker disease

摘要:

运用PCR方法扩增利用核糖体展示技术筛选的抗柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,XAC)的单链抗体(ScFv95)基因片段,将单链抗体基因重组到原核表达载体pET30a(+)中,构建单链抗体高效表达载体pET30a(+)-XAC-ScFv。再将pET30a(+)-XAC-ScFv质粒转化进大肠杆菌BL21(DE3)后诱导表达,并对表达产物进行纯化、复性及活性检测。获得了抗XAC单链抗体的高效表达蛋白,以包涵体形式存在的表达蛋白大小约32kDa。包涵体蛋白经过变性、纯化和复性后,初步获得有功能的单链抗体。同时用Biacore分析XAC-ScFv-95与XAC LPS作用,结果表明复性后的XAC-ScFv-95具有较高的亲和力,从而为柑桔溃疡病菌XAC的免疫诊断和防治研究提供了新的工具和途径。

Abstract:

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.