Beclin1进化保守区-SA融合蛋白偶联生物素-A10适配子制备PSMA靶向系统及其对前列腺癌细胞的抑制

Construction of PSMA targeting system with fusion protein of evolutionary conserved district of Beclin1-streptavidin coupling biotin-aptamer A10 and inhibition effect of the targeting system on prostatic cancer cells

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 fusion protein prokaryotic expression vector of streptavidin（ SA） and evolutionary conserved district（ ECD） of Beclin1,to prepare PSMA targeting system the fusion protein coupling specific aptamer A10 of biotin-prostate specific membrane antigen（ PSMA） and to identify its promotion effect on apoptosis and autophagy activities of PSMA＋prostatic cancer cells. Methods： Fusion SA-ECD gene was obtained by gene synthesis technology and then cloned into vector PUC57; after confirmation of correct synthesis,the SA-ECD gene was directly inserted into the prokaryotic expression vector PET30a; the fusion protein was expressed with inducement of IPTG,purified with Ni affinity gel chromatography column and identified with Western blotting. PSMA specific conjugate of PSMA,namely the targeting system,was prepared by mixing the biotin-A10 and the SA-ECD at the molar ratio of 4∶ 1; Combination of the biotin-A10 and the SA-ECD was confirmed by electrophoretic mobility shift assay（ EMSA）. Effects of the targeting system on apoptosis,expression of autophagy related proteins and viability of the prostatic cancer cells were detected by flow cytometry,Western blotting and trypan blue assays,respectively. Results： Double restriction enzyme digestion and gene sequencing assays verified that the recombinant plasmid PET30a-SA-ECD was successfully constructed; the SA-ECD fusion protein was efficiently expressed in E. coli with induction of IPTG and successfully purified by affinity gel chromatography. EMSA confirmed that SA-ECD could effectively bind biotin-A10. The targeting system could promote apoptosis and autophagy of the prostatic cancer cells,and inhibit its viability. Conclusion： The SA-ECD fusion protein was successfully expressed and purified,and the targeting system of biotin-A10 and SA-ECD could kill the PSMA＋prostatic cancer cells.