基于酶催化抗体药物偶联物中抗体的定点突变及鉴定

发布时间：2018-04-08 08:14

本文选题：抗体　切入点：定点突变　出处：《药学学报》2017年03期

【摘要】：谷氨酰胺转移酶催化谷氨酰胺与赖氨酸及其衍生物形成异肽键,可用于抗体与小分子药物的定点偶联。本文在构建的抗人CD24嵌合抗体c G7的基础上,定点突变c G7获得具有4个可催化的谷氨酰胺位点的嵌合抗体c G7Q。本研究采用overlap PCR技术将抗体重链CH段第297位天冬酰胺突变成谷氨酰胺,构建含突变重链的真核表达载体。将含突变重链与轻链的载体共转染CHO-s细胞,筛选稳定高产单克隆细胞株,表达及鉴定目的蛋白c G7Q,表面等离子共振法与流式细胞术分析c G7Q与人CD24分子的结合能力,乳酸脱氢酶释放实验鉴定定点突变对抗体依赖细胞介导的细胞毒作用(ADCC)的影响,结果显示,经改造的c G7Q保持母体单抗的结构和特异性结合能力及部分ADCC效应,为后续制备靶向CD24定点偶联药物奠定基础。
[Abstract]:Glutamine transferase catalyzes the formation of isopeptide bonds between glutamine and lysine and its derivatives.Based on the construction of anti-human CD24 chimeric antibody c G7, the chimeric antibody c G7Q with four catalytic glutamine sites was obtained by site-directed mutagenesis.In this study, overlap PCR technique was used to construct eukaryotic expression vector containing mutated heavy chain by mutating asparagine into glutamine at the 297th position of Ch segment of antibody heavy chain.The vector containing mutated heavy chain and light chain was co-transfected into CHO-s cells, and stable and high-yielding monoclonal cell lines were screened. The target protein c G7Qwas expressed and identified. The binding ability of c G7Q to human CD24 molecule was analyzed by surface plasmon resonance and flow cytometry.The effect of site-directed mutagenesis on the cytotoxicity mediated by antibody dependent cells was identified by lactate dehydrogenase release assay. The results showed that the modified c G7Q maintained the structure, specific binding ability and partial ADCC effect of the mAb.It lays a foundation for the further preparation of targeted CD24 conjugated drugs.
【作者单位】：中国药科大学生命科学与技术学院;浙江省中医院;
【基金】：国家自然科学基金面上项目资助(81473125) 江苏省自然科学基金项目资助(BK20161459) 华海药业研究生创新基金(1010040003) 江苏省研究生科研创新计划(硕士)(KYZZ15_0190) 大学生创新实验训练项目基金(SY15090)
【分类号】：R945

【相似文献】