迟缓爱德华菌抗独特型单链抗体基因的构建、表达及鉴定

Construction,expression and identification of the anti-idiotypic single chain variable fragment against Edwardsiella tarda

目的构建、表达及鉴定迟缓爱德华菌抗独特型单链抗体基因。方法采用RT-PCR方法从分泌迟缓爱德华菌独特型单克隆抗体的杂交瘤细胞株(1E11)中克隆出VH和VL可变区基因,再通过重叠延伸拼接PCR方法在VH和VL可变区基因之间引入连接肽(Gly4Ser)3,体外构建单链抗体基因;将其克隆至表达载体pET-28a并在大肠杆菌中表达,表达产物纯化后,用SDS-PAGE、Western blot及ELASA进行鉴定。结果迟缓爱德华菌抗独特型单链抗体基因scFv在BL21(DE3)菌中获得表达,表达产物以不溶性包涵体形式存在,经过溶解包涵体、纯化和体外复性,获得了高纯度的单链抗体片段,重组蛋白的分子质量为27 ku。ELASA分析结果证实迟缓爱德华菌抗独特型单链抗体基因scFv与原代抗体一样,具有较高的抗原亲和力。结论成功构建了迟缓爱德华菌抗独特型单链抗体基因scFv,为进一步制备迟缓爱德华菌抗独特型抗体基因工程疫苗奠定基础。

Objective To construct, express and identify the anti-idiotypic antibody single chain variable fragment （scFv） against Edwardsiella tarda. Methods By using RT-PCR method, the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody （mAb） 1Ell against Edwardsiella tarda were cloned and joined with a （Gly4 Ser）3 linker, and the scFv in the orientation of VL-linker-VH was constructed. It was then cloned into vector plasmid pET-28a, expressed in Escherichia cell BL21 （DE3）, and confirmed by SDS-PAGE, Western blot and ELISA. Results The recombinant scFv could be expressed in E. coli BL21 （DE3） in a fusion protein pattern. The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded. The SDS-PAGE and Western blot analysis showed that the molecular weight of scFv protein was 27 ku. Indirect ELISA confirmed that the scFv had the binding activity to the antigen. Conolusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E. coli BL21 （DE3）, providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda .