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目的:构建链霉亲和素(streptavidin,SA)与Beclin1进化保守区(evolutionary conserved district,ECD)融合蛋白原核表达载体,将融合蛋白与生物素-前列腺特异性膜抗原(prostate specific membrane antigen,PSMA)特异性适配子A10偶联制备PSMA靶向系统,鉴定其促进PSMA~+前列腺癌细胞凋亡及自噬的功能。方法:采用基因合成技术获得融合基因SA-ECD,克隆至PUC57载体鉴定无误后,再将其定向插入原核表达载体PET30a,IPTG诱导融合蛋白表达,镍亲和凝胶层析柱纯化融合蛋白,Western blotting鉴定。按照生物素-A10∶SA-ECD摩尔比为4∶1的比例制备PSMA特异性的偶联物即靶向系统,凝胶迁移阻滞实验(electrophoretic mobility shift assay,EMSA)验证SA-ECD与生物素的结合,流式细胞术、Western blotting、锥虫蓝染色分别检测靶向系统对前列腺癌细胞凋亡、自噬相关蛋白表达和细胞活力的影响。结果:双酶切及基因测序证实重组质粒PET30a-SA-ECD构建成功,IPTG诱导SA-ECD融合蛋白在大肠杆菌中获得高效表达并经亲和凝胶层析成功纯化。EMSA实验证明SA-ECD能有效结合生物素-A10。靶向系统可促进PSMA~+前列腺癌细胞凋亡和自噬,同时抑制癌细胞活力。结论:成功表达并纯化融合蛋白SA-ECD,构建的生物素-A10∶SA-ECD靶向系统能够杀伤PSMA~+前列腺癌细胞。
OBJECTIVE: To construct a prokaryotic expression vector for streptavidin (SA) and Beclin1 evolutionary conserved district (ECD) fusion protein. The fusion protein was fused with the biotin-prostate specific membrane antigen (PSMA ) -specific aptamer A10 was used to prepare PSMA targeting system to identify its function of promoting apoptosis and autophagy of PSMA ~ + prostate cancer cells. Methods: The fusion gene SA-ECD was obtained by gene synthesis and cloned into PUC57 vector. After being inserted into prokaryotic expression vector PET30a, the fusion protein was induced by IPTG. The fusion protein was purified by nickel affinity gel chromatography. blotting identification. PSMA-specific conjugates, targeting systems, were prepared at a 4: 1 molar ratio of biotin-A10: SA-ECD. Electrophoretic mobility shift assay (EMSA) Western blotting and Trypan blue staining were used to detect the effect of targeting system on apoptosis, autophagy-related protein expression and cell viability in prostate cancer cells. Results: Double enzyme digestion and gene sequencing confirmed that the recombinant plasmid PET30a-SA-ECD was successfully constructed. The IPTG-induced SA-ECD fusion protein was highly expressed in E. coli and successfully purified by affinity gel chromatography. EMSA experiments show that SA-ECD can bind biotin-A10 effectively. Targeting system can promote PSMA ~ + prostate cancer cell apoptosis and autophagy, while inhibiting cancer cell viability. CONCLUSION: The biotin-A10: SA-ECD targeting system can successfully kill PSMA ~ + prostate cancer cells by successfully expressing and purifying the fusion protein SA-ECD.