毕赤酵母表达CD63单链抗体及其生物活性的鉴定

Expression and biological characterization of anti-CD63 single-chain variable fragment antibody in Pichia pastoris

摘要:

为了制备能够特异性识别CD63蛋白胞外大环中保守结构域的抗体，本研究利用毕赤酵母分泌表达CD63单链抗体(single chain variable fragment,scFv)，纯化后得到能特异性结合CD63蛋白以及SK-MEL-28细胞表面CD63分子的CD63 scFv。对筛选得到的CD63单克隆抗体细胞株进行可变区测序得到重链可变区(variable heavy chain,VH)与轻链可变区(variable light chain,VL)，经柔性肽连接构成scFv，通过密码子优化后全基因合成CD63 scFv序列并克隆至毕赤酵母表达质粒pPICZα-A，质粒经Sac I线性化后电转到毕赤酵母X33，1%甲醇诱导表达scFv，发酵上清通过Ni柱纯化，并用SDS-PAGE和Western blotting进行了鉴定。通过免疫印迹、免疫荧光、基于细胞的酶联免疫吸附实验、流式细胞术对CD63 scFv的生物活性进行鉴定。本研究成功构建了能够分泌表达抗CD63 scFv的毕赤酵母菌株，抗体分子量约为30 kDa，能与CD63蛋白特异性结合。抗CD63 scFv在毕赤酵母中的表达是一种经济有效的抗体获取方法，为大规模生产抗CD63抗体奠定了基础。

Abstract:

To prepare antibodies that specifically recognize the conserved domain in the large extracellular loop of the CD63 protein, we expressed anti-CD63 single-chain variable fragment (scFv) antibody in Pichia pastoris in a secreted form. The purified expression product was found to bind specifically with CD63 protein and recognize CD63 on the surface of SK-MEL-28 cells. The variable region of the anti-CD63 monoclonal antibody in an anti-CD63-positive cell line was sequenced. The anti-CD63 scFv consisted of a variable heavy chain and a variable light chain linked by a flexible peptide was then designed. After codon optimization, the gene was synthesized and cloned into the expression plasmid pPICZα-A. The SacI-linearized plasmid was electroporated into P. pastoris X33, and 1% methanol were used to induce the expression of scFv. The fermentation supernatant was purified by Ni column. Anti-CD63 scFv was identified by SDS-PAGE and Western blotting, and its biological activities were analyzed by immunoblotting, immunofluorescence, cell-based ELISA, and flow cytometry. A P. pastoris strain capable of expressing and secreting anti-CD63 scFv was successfully obtained. The antibody had a molecular weight of approximately 30 kDa and specifically recognized CD63 protein. The expression of anti-CD63 scFv in P. pastoris paves the way for the production of anti-CD63 antibodies on a large-scale, which is undoubtedly an economical and effective way of antibody acquisition.