迟缓爱德华菌抗独特型单链抗体基因的构建、表达及鉴定

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V_H和V_L可变区基因, 再通过重叠延伸拼接PCR方法在V_H和V_L可变区基因之间引入连接肽(Gly_4ser)_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.

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

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) 1E11 against Edwardsiella tarda were cloned and joined with a (Gly_4ser)_3 linker, and the scFv in the orientation of V_L-linker-V_H was constructed. It was then cloned into vector plasmid pET-28a, expressed in Escherichia coli 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 had the binding activity to the antigen. Conclusion 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.