靶向PSMA多价纳米抗体的制备及其生物学活性表征  

Development and Biological Activity Analysis of PSMA Specific Mutivalent Nanobodies

作　　者：鲍奕恺 洪皓飞 施杰 周志昉 吴志猛 BAO Yi-kai;HONG Hao-fei;SHI Jie;ZHOU Zhi-fang;WU Zhi-meng(Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院糖化学与生物技术教育部重点实验室,无锡214122

出　　处：《中国生物工程杂志》2022年第5期37-45,共9页China Biotechnology

基　　金：国家自然科学基金(32000904、21907038、22177040);江苏省自然科学基金(BK20200601);中国博士后科学基金(2021M691293)资助项目。

摘　　要：目的:构建原核表达系统,制备靶向前列腺特异性膜抗原(prostate-specific membrane antigen,PSMA)多价纳米抗体并初步评价其生物学活性。方法:Bglbrick法构建多价纳米抗体表达载体,转化至大肠杆菌表达并利用亲和层析法纯化。联合蛋白质电泳和Western blot验证纯化产物,BCA法检测表达量。通过免疫荧光和流式细胞术定性评估PSMA特异性亲和能力,细胞ELISA法定量检测PSMA亲和水平,流式细胞术检测内吞效率。结果:成功构建靶向PSMA单价、二价、三价和四价纳米抗体大肠杆菌表达菌株。发酵结果表明四种纳米抗体均能在摇瓶水平实现高效可溶表达,其中二价纳米抗体表达量最高[(259.14±23.56)mg/L],单价纳米抗体表达量最低[(100.58±6.27)mg/L]。亲和实验结果证实四种纳米抗体均能特异性识别并结合PSMA阳性肿瘤细胞,与单价纳米抗体相比,二价、三价和四价纳米抗体对PSMA亲和能力分别提高了3.32倍、2.29倍和2.03倍。最后的内吞实验显示四种纳米抗体均能被PSMA阳性肿瘤细胞高效摄取,30 min内的摄取率均在80%以上。结论:靶向PSMA的多价纳米抗体,尤其是二价纳米抗体,具有比单价纳米抗体更高的产量和亲和水平,且具备不亚于单价纳米抗体的内吞效率,是未来基于PSMA肿瘤诊疗试剂开发的重要候选。Objective:To express PSMA(prostate-specific membrane antigen)specific multivalent nanobodies in the prokaryotic system,and preliminarily evaluate their biological properties.Methods:The multivalent nanobody expression vectors were constructed by the Bglbrick method,and then transferred into E.coli Rosetta(DE3)for protein production.SDS-PAGE and Western blot assays were performed to identify the purified nanobody samples.BCA(bicinchoninic acid assay)kits were used for expression yield determination.The specific binding affinities were evaluated by immunofluorescence and flow cytometry.The binding EC;values were detected by cell-based ELISA.The endocytosis efficiency was investigated via flow cytometry.Results:Recombinant E.coli strains for PSMA specific monovalent,bivalent,trivalent and tetravalent nanobodies production were successfully constructed,respectively.Fermentation results showed that all four kinds of nanobodies could be expressed as soluble proteins with high yields at shake flask level,among which the bivalent nanobody presented the highest expression yield[(259.14±23.56)mg/L],whereas the monovalent type lowest[(100.58±6.27)mg/L].In the subsequent cell binding assays,results indicated that all four nanobody samples could specifically recognize and bind to PSMA-positive tumor cells.Notably,compared with monovalent nanobody,the binding affinity of bivalent,trivalent and tetravalent nanobodies were improved approximately 3.32-,2.29-and 2.03-fold,respectively.Finally,the endocytosis experiments were conducted and the results suggested that all four kinds of nanobodies could be efficiently endocytosed by PSMA-positive tumor cells and the uptake rates in 0.5 h were all above 80%.Conclusion:PSMA specific multivalent nanobodies,especially PSMA specific bivalent nanobody,had higher expression yield and better binding affinity than monovalent nanobody.Meanwhile,they remained the same uptake level with monovalent nanobody.In this context,PSMA specific multivalent nanobodies could be a potential class of candid

关 键 词：前列腺癌 前列腺特异性膜抗原(PSMA) 多价纳米抗体 

分 类 号：Q816[生物学—生物工程]