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目的:提高抗肝癌靶向超抗原SEA(D227A)的产量和稳定性,构建单链二硫键稳定抗体靶向超抗原分子scdsFv-SEA(D227A)。方法:构建scdsFv SEA(D227A)表达质粒,用IPTG诱导其在大肠杆菌BL21plusS表达。包涵体经洗涤和复性后,用QSepharoseHP和Hiprep26/60SephacrylS-200HR纯化。纯化蛋白经AMS烷化处理并结合PAGE电泳来检测二硫键形成情况,并通过ELISA和MTS分别检测重组蛋白与肝癌细胞的结合活性、稳定性和细胞杀伤活性。结果:重组蛋白以包涵体的形式表达,表达量占菌体总蛋白的30%以上。通过复性及QSepharoseHP离子交换柱和Hiprep26/60SephacrylS-200H凝胶过滤两步纯化后,获得目的蛋白,每升诱导菌液的产量高达60mg。活性测定结果表明,相比与单链抗体靶向超抗原和二硫键抗体靶向超抗原SEA,二硫键抗体靶向超抗原在不影响结合活性和杀伤效果的情况下,稳定性有了较大提高。结论:建立了一种新的稳定的免疫毒素和提高其产量的方法,为hscFv25抗体靶向超抗原的临床应用奠定了基础,同时为其他低稳定性或低产量抗体的改造提供了借鉴的方法。
OBJECTIVE: To improve the yield and stability of the targeted superantigen SEA (D227A) against hepatocellular carcinoma (HCC) and to construct scdsFv-SEA (D227A), a single chain disulfide stabilized antibody targeting superantigen. Methods: The scdsFv SEA (D227A) expression plasmid was constructed and induced by IPTG in E. coli BL21plusS. Inclusion bodies were washed and refolded and purified with QSepharose HP and Hiprep 26/60 Sephacryl S-200 HR. The purified protein was subjected to AMS alkylation and PAGE electrophoresis to detect disulfide bond formation. The binding activity, stability and cytotoxic activity of the recombinant protein to the hepatoma cells were detected by ELISA and MTS respectively. Results: The recombinant protein expressed in the form of inclusion bodies, the expression amount accounted for more than 30% of the total bacterial protein. Purification by renaturation and QSepharoseHP ion exchange column and Hiprep26 / 60SephacrylS-200H gel filtration after purification, the desired protein was obtained, the yield per liter of induced bacterial liquid up to 60mg. The results of the activity assay showed that the stability of the disulfide antibody-targeted superantigen without any effect on the binding activity and killing effect compared to that of the single-chain antibody-targeting superantigen and disulfide antibody targeting superantigen SEA Larger increase. CONCLUSION: A new stable immunotoxin and its method of production are established, which lays the foundation for the clinical application of hscFv25 antibody targeting superantigen and provides a reference method for the modification of other low-stability or low-yield antibodies .