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，Annexin Ⅴ-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56 000。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；25 μg/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 Annexin Ⅴ-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 56 000. The purified Nb 2B3-PE38 could specifically bind to the recombinant CD70 antigen and the CD70 molecule on the surface of 786-O cells. 25 μg/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.

国家自然科学基金（No. 31570935）