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

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.