农业生物技术学报2011，Vol.19Issue(1)：178-185,8.DOI:10.3969/j.issn.1674-7968.2011.01.026
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
Reconstruction, Expression and Characterization of Disulfide-stabilized Single Chain Variable Fragment to Xanthomonas axonopodis pv. citri
摘要
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 ％ aider 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.关键词
柑橘溃疡病菌/基因改造/sc-dsFv重组抗体/复性/BIAcore分析/原核表达Key words
Xanthomonas axonopodis pv. citri/ Genetic modification/ Sc-dsFv recombinant antibody/ Renaturation/BIAcore analysis/ Prokaryote expression引用本文复制引用
殷幼平,李蒙,于红,吴瑜佳,王中康..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[J].农业生物技术学报,2011,19(1):178-185,8.基金项目
本研究由"十一五"国家科技支撑计划项目(NO.2007BAD47B03-5)资助 （NO.2007BAD47B03-5）
