CAJ | 학술논문

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.
목적 구건、표체급감정지완애덕화균항독특형단련항체기인.방법 채용RT-PCR방법종분비지완애덕화균독특형단극륭항체적잡교류세포주(1E11)중극륭출V_H화V_L가변구기인, 재통과중첩연신병접PCR방법재V_H화V_L가변구기인지간인입련접태(Gly_4ser)_3,체외구건단련항체기인;장기극륭지표체재체pET-28a병재대장간균중표체,표체산물순화후,용SDS- PAGE、Western blot급ELASA진행감정.결과 지완애덕화균항독특형단련항체기인scFv재BL21(DE3)균중획득표체,표체산물이불용성포함체형식존재,경과용해포함체、순화화체외복성,획득료고순도적단련항체편단,중조단백적분자질량위27 ku.ELASA분석결과증실지완애덕화균항독특형단련항체기인scFv여원대항체일양,구유교고적항원친화력.결론 성공구건료지완애덕화균항독특형단련항체기인scFv,위진일보제비지완애덕화균항독특형항체기인공정역묘전정기출.
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.