Abstract：Citrus canker is a severe bacterial disease of most commercial citrus species and cultivars around the world. The purpose of this study is to construct specificity and stability antibody for development rapid and reliable procedures for diagnosis and control of this pathogen. To construct the recombinant mouse anti-Xac disulfide-stabilized single chain variable fragment (sc-dsFv), we used mouse anti-Xac disulfide-stabilized as templates for genetic modification, amplified the genes of variable region of heavy chain(VH) and variable region of light chain(VL), and introduced a sequence encoding 5'-terminal 12 amino acid of heavy chain constant region CH1, as a linker, between VH and VL by overlap extension PCR. The recombinant gene was then cloned into expression plasmid pET24a(+),transformed and expressed into Escherichia coli BL21 (DE3). The products of expression were renatured and analyzed by SDS-PAGE and Western blot. The affinity of sc-dsFv to Xac lipopolysaccharides(LPS) was tested by BIAcore, the specificity and stability were detected by ELISA and Dot blot. The results showed that the linker sequence was introduced into VH and VL successfully, and the recombinant gene expressed at high level in E. coli BL21(DE3). The most of the express protein existed in the form of inclusion body. Purity of the protein was higher than 90 % after purified by Ni-NTA and renaturaton in vitro. Dot blot, ELISA and BIAcore determination demonstrated that sc-dsFv could bind antigen specificity and was more stable than that of sigle-chain variable fragment(scFv), that means the expression products were refolded correctly after renaturation. The expression yield of the antibody was higher than that of dsFv in the same conditions. Our study approved that a heavy chain constant region CH1 introduced between VH and VL genes of anti-Xac can improve the affinity, specificity and stability of expressive antibody. It will be useful for X. axonopodis pv. citri intuitive and rapid diagnosis.
