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优化以包涵体形式表达的抗Ⅳ型胶原酶单链抗体与力达霉素辅基蛋白融合蛋白Fv-LDP制备过程。通过单因素或正交设计优化其包涵体制备与溶解、固定化金属螯合层析和分步透析复性过程,Sephadex G-75层析精细纯化蛋白,基于流式细胞术的饱和结合实验检测其抗原结合活性。优化后,Fv-LDP包涵体纯度为63.9%,溶解度为95.7%;纯化后Fv-LDP纯度达95%以上,复性后单体含量占60%,精细纯化后提升为85%,比初始复性条件提高5.6倍,能够与人肺腺癌PAa细胞和肝癌BEL-7402细胞结合。Fv-LDP制备过程被成功优化,为其生产和研发奠定了实验基础,也为其他以包涵体形式表达的基于单链抗体的小型化抗体药物制备提供了比较实用的方法。
The preparation of Fv-LDP, an anti-type IV collagenase single chain antibody expressed in inclusion bodies, was optimized with lidamycin-based protein fusion protein. The inclusion body preparation and dissolution, immobilized metal chelate chromatography and fractional dialysis refolding were optimized by single factor or orthogonal design. Sephadex G-75 chromatography purified protein was purified by flow cytometry based on saturation binding assay Its antigen binding activity. After purification, the purity of Fv-LDP inclusion body was 63.9%, and the solubility was 95.7%. The purity of purified Fv-LDP was over 95%. After refolding, the content of monomer was 60% and the purity of Fv-LDP was 85% Sexual conditions increased 5.6-fold, with human lung adenocarcinoma PAa cells and liver cancer BEL-7402 cells. The preparation of Fv-LDP was successfully optimized, which laid the experimental foundation for its production and research and development. It also provided a more practical method for the preparation of other small antibody based on single chain antibody expressed in the form of inclusion body.