Reconstruction, expression and characterization of disulfide-stabilized single chain variable fragment to Xanthomonas axonopodis pv. Citri | 柑橘溃疡病菌单链二硫键稳定重组抗体的构建、表达和特性

Chinese. 柑橘溃疡病(citrus bacterial canker disease，CBCD)是危害柑橘(Citrus reticulata Blanco)的重要病害，为了给柑橘溃疡病的现场快速诊断提供一种稳定的重组抗体，构建一种快速检测技术，本研究以鼠源抗柑橘溃疡病菌(Xanthomonas axonopodis pv. citri, Xac)表面脂多糖的二硫键稳定性抗体dsFv为模板进行基因改造，利用重叠延伸PCR在其重链可变区(VH)和轻链可变区(VL)之间引入一条人类抗体重链恒定区1(CH1)5'端12个氨基酸的序列作为连接肽，构建重组单链二硫键稳定抗体基因sc-dsFv。将获得的基因连接pET24a(+)载体，转入大肠杆菌(Escherichia coli) BL21(DE3)得到工程菌，IPTG诱导表达。表达产物经体外复性并以Ni-NTA亲和层析对目标蛋白sc-dsFv进行纯化，利用斑点免疫杂交、ELISA和BIAcore检测抗体的活性、稳定性、特异性及亲和力。构建的抗体基因经测序结果表明，抗体重链可变区(VH)和轻链可变区(VL)基因之间成功引入目标连接肽序列，重组sc-dsFv蛋白实现了原核高效表达；通过体外复性和Ni-NTA柱纯化获得纯度高于90%的sc-dsFv蛋白。特异性和亲和性测定结果表明，该重组抗体具有特异的抗原结合活性和良好的抗原亲和力，与本实验室保存的抗柑橘溃疡病菌表面脂多糖scFv及dsFv比较，产量有明显提高且稳定性也有所提高。这种具有特异抗原结合活性的稳定抗体sc-dsFv的获得为柑橘溃疡病的快速免疫诊断提供了高效优质重组抗体，并为病害治疗性抗体制备提供了有效的方法。

English. 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.