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Archive > Volume 30 Issue 8 > 2023,30(8):665-671. DOI:10.3872/j.issn.1007-385X.2023.08.003 Prev Next
Basic Research
Preparetion of anti-CD70 nanobody immunotoxin fused with PE38 and its killing effect on renal clear cell carcinoma 786-O cells
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Abstract:
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.
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