抗人肺腺癌单链抗体的构建及在大肠杆菌中的表达

目的:构建抗肺腺癌的单链抗体(scFv),研究其表达条件,为将这一小分子抗体应用于临床奠定基础.方法:将抗人肺腺癌单克隆抗体的重链及轻链可变区基因插入表达载体pFUW80、pTH、pTF,分别在大肠杆菌XL1-Blue、Top10、GI698/GI724中诱导表达,得到噬菌体抗体或包涵体.用SDS-PAGE和ELISA鉴定检测活性.结果:SDS-PAGE的结果表明,在pTH和pTF的表达产物中,有一条43kD的特异条带,其分子量与预期相符,单链抗体以包涵体形式出现,其表达量达细菌总蛋白的18.6%.ELISA的结果表明,噬菌体抗体和经复性处理的包涵体具有与亲本单抗相同的特异性.结论:成功地构建和在大肠杆菌中表达了抗肺腺癌单链抗体.

Construction and Expression in E. coli of scFv against Human Lung Adenocarcinoma

To construct a single-chain antibody (scFv) against lung adenocarcinoma and research its expressing conditions for establishing a clinical base. Methods: The variable genes of McAb anti-human lung adenocarcinoma, which were cloned from hybridoma cell WLA-2C4, were inserted into phagemid pFUW80 and expressed scFv in XLl-Blue. Also the scFv genes were inserted into another two vectors pTH and pTF. The corresponding host strains were ToplO and GI724/GI698, the inducing agents were EPTG and tryptophan, respectively. SDS-PAGE and ELBA methods were used to assay the expression products-phage antibodies in XLl-Blue and inclusion bodies in ToplO and GI724/ GI698. Results: The scFv had the same specificity of the original monoclonal antibody against the antigen of A549 cell. The inclusion bodies were obtained at high yield 18.6% of the total E. coli proteins. Conclusion: A scFv against lung adenocarcinoma was successfully constructed and expressed in E. coli.