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目的构建人源性抗前列腺特异性膜抗原(PSMA)单链抗体(sc Fv)的原核表达载体,在大肠杆菌中诱导表达并纯化后,对其生物学活性进行鉴定。方法将人源性抗PSMA sc Fv的基因克隆入原核表达载体p ET302,将其转化入大肠杆菌BL21后用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,摸索不同诱导条件使sc Fv呈可溶性表达,经SDS-PAGE和Western blot法确认表达后,利用Ni2+-NTA亲和层析柱纯化目的蛋白。利用流式细胞术检测sc Fv与PSMA阳性和阴性细胞的结合情况。结果成功构建人源性抗PSMA sc Fv的原核表达载体;在30℃、IPTG终浓度为0.05 mmol/L时,可实现目的蛋白的可溶性表达;经Ni2+-NTA亲和层析柱纯化后获得纯度较高的抗PSMA sc Fv蛋白;流式细胞术结果显示该sc Fv可与PSMA阳性的LNCa P前列腺癌细胞特异性结合,而不与PSMA阴性的PC-3细胞结合。结论在大肠杆菌成功表达了可溶性人源性抗PSMA sc Fv,获得的抗PSMA sc Fv能够特异性地与PSMA阳性前列腺癌细胞结合。
Objective To construct a prokaryotic expression vector of scFv against human prostate specific membrane antigen (PSMA) and express it in Escherichia coli before its biological activity was identified. Methods The human anti-PSMA sc Fv gene was cloned into the prokaryotic expression vector p ET302, which was transformed into E. coli BL21 and induced with IPTG The expression of sc Fv was induced by different conditions. After the expression was confirmed by SDS-PAGE and Western blot, the target protein was purified by Ni2 + -NTA affinity chromatography. Flow cytometry was used to detect the binding of sc Fv to PSMA positive and negative cells. Results The prokaryotic expression vector of human anti-PSMA sc Fv was successfully constructed. Soluble expression of the target protein was achieved at a final IPTG concentration of 0.05 mmol / L at 30 ℃. After purification by Ni2 + -NTA affinity chromatography, the purity Higher anti-PSMA sc Fv protein; flow cytometry results show that this sc Fv specifically binds PSMA-positive LNCa P prostate cancer cells without binding to PSMA-negative PC-3 cells. Conclusions Soluble human-derived anti-PSMA sc Fv was successfully expressed in E. coli and the obtained anti-PSMA sc Fv was able to bind specifically to PSMA-positive prostate cancer cells.