融合PE38的抗CD70纳米抗体免疫毒素的制备及其对肾透明细胞癌786-O细胞的杀伤作用  

Preparetion of anti-CD70 nanobody immunotoxin fused with PE38 and its killing effect on renal clear cell carcinoma 786-O cells

作　　者：徐新兰 刘畅 张鑫 胡倩倩 李江伟[1] XU Xinlan;LIU Chang;ZHANG Xin;HU Qianqian;LI Jiangwei(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046,Xinjiang,China)

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源基因工程重点实验室,新疆乌鲁木齐830046

出　　处：《中国肿瘤生物治疗杂志》2023年第8期665-671,共7页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金(No.31570935)。

摘　　要：目的:构建靶向CD70分子的重组免疫毒素,通过表达、纯化制备PE38与抗CD70纳米抗体重组蛋白,体外抗肿瘤实验探究重组蛋白是否对高表达CD70分子的阳性肿瘤细胞具有杀伤活性。方法:通过基因工程手段,将CD70纳米抗体Nb 2B3基因片段通过一个连接子与pET21a-PE38基因片段相连,获得重组表达载体pET21a-Nb 2B3-PE38并转入BL21(DE3)感受态细胞中进行表达、纯化与鉴定。用间接ELISA及FACS法检测Nb 2B3-PE38与CD70分子的结合活性,MTT法检测Nb 2B3-PE38对高表达CD70分子的肾透明细胞癌786-O细胞的体外杀伤活性,AnnexinⅤ-FITC/PI双染法检测Nb 2B3-PE38对786-O细胞凋亡的影响。结果:成功构建抗CD70纳米抗体重组免疫毒素Nb 2B3-PE38,纯化获得纯度>90%的重组蛋白,SDS-PAGE及WB检测结果表明目的蛋白正确表达,分子量为56000。纯化后的Nb 2B3-PE38能与重组CD70抗原及786-O细胞表面的CD70分子特异性结合;25μg/mL Nb 2B3-PE38即对786-O细胞产生极显著的杀伤作用(P<0.001),并且促进786-O细胞的细胞凋亡(P<0.01),其杀伤效应强于阳性对照顺铂(P<0.01)。结论:成功制备了特异性靶向CD70分子的免疫毒素Nb 2B3-PE38,其能够有效杀伤786-O细胞并诱导细胞凋亡且效果强于顺铂。Objective:To construct a recombinant immunotoxin targeting CD70 molecule,prepare PE38 and anti-CD70 nanobody recombinant proteins by expression and purification,and explore whether the recombinant proteins have killing activity on positive tumor cells with high expression of CD70 molecule through in vitro anti-tumor experiments.Methods:The CD70 nanobody Nb 2B3 gene fragment was ligated with the pET21a-PE38 gene fragment through a linker by genetic engineering,and the recombinant expression vector pET21a-Nb 2B3-PE38 was obtained and transformed into BL21(DE3)competent cells for expression purification and identification.The binding activity of Nb 2B3-PE38 to CD70 molecule was detected by indirect ELISA and FACS.The in vitro killing activity of Nb 2B3-PE38 against renal clear cell carcinoma(786-O)with high expression of CD70 molecule was detected by MTT assay.The effect of Nb 2B3-PE38 on the apoptosis of 786-O cells was identified by AnnexinⅤ-FITC/PI double staining.Results:The recombinant immunotoxin Nb 2B3-PE38 against CD70 nanobody was successfully constructed,and the recombinant protein with a purity higher than 90%was purified.The results of SDS-PAGE and WB showed that the target protein was correctly expressed and the molecular weight was 56000.The purified Nb 2B3-PE38 could specifically bind to the recombinant CD70 antigen and the CD70 molecule on the surface of 786-O cells.25μg/mL Nb 2B3-PE38 has a highly significant killing effect on 786-O cells(P<0.001),and promotes apoptosis of 786-O cells(P<0.01).Its killing effect was stronger than that of positive control cisplatin(P<0.01).Conclusion:The immunotoxin Nb 2B3-PE38,which specifically targets CD70 molecules,was successfully prepared.This immunotoxin can effectively kill 786-O cells and induce apoptosis.

关 键 词：CD70 铜绿假单胞杆菌外毒素 纳米抗体 PE38 重组免疫毒素 肾透明细胞癌 786-O细胞 细胞毒性 靶向治疗 

分 类 号：R979.1[医药卫生—药品] R730.54[医药卫生—药学] R737.11