论文部分内容阅读
目的:构建3TAT-DRBD重组载体,表达和纯化融合蛋白,并对其siRNA结合活性和穿膜功能进行初步验证。方法:采用基因合成技术获取靶基因3TAT-DRBD,并克隆到原核表达载体pET-44b中;用IPTG诱导融合蛋白表达,镍亲和凝胶层析柱纯化融合蛋白,凝血酶切除标签,Western blotting鉴定。凝胶迁移阻滞实验验证DRBD和siRNA的结合能力,激光共聚焦显微镜观察TAT的穿膜能力。结果:限制性酶切和基因测序表明重组质粒pET-44b-3TAT-DRBD构建成功;IPTG诱导后3TAT-DRBD融合蛋白(含Nus标签和S标签)在大肠杆菌中高效表达,可溶性蛋白占菌体总蛋白约80%;成功切除融合标签并纯化了无标签的融合蛋白,经Western blotting鉴定其相对分子质量约为17 000;凝胶迁移阻滞实验证明,融合蛋白3TAT-DRBD能有效结合靶向survivin基因的siRNA(survivin-siRNA);激光共聚焦显微镜下可见,在TAT的介导下survivin-siRNA穿透胞膜进入前列腺癌PC3细胞的效率明显增高。结论:成功表达并纯化了具有siRNA结合活性与穿膜功能的3TAT-DRBD融合蛋白,为进一步3TAT-DRBD的功能研究及临床应用奠定了基础。
OBJECTIVE: To construct 3TAT-DRBD recombinant vector, express and purify the fusion protein, and to verify its binding activity and transmembrane function. Methods: The target gene 3TAT-DRBD was obtained by gene synthesis and cloned into the prokaryotic expression vector pET-44b. The fusion protein was induced by IPTG. The fusion protein, the thrombin removal tag, the Western blot blotting identification. The binding ability of DRBD and siRNA was verified by gel migration retardation assay, and the transmembrane capacity of TAT was observed by laser confocal microscopy. Results: Restriction enzyme digestion and gene sequencing showed that the recombinant plasmid pET-44b-3TAT-DRBD was constructed successfully. After induced by IPTG, the 3TAT-DRBD fusion protein (containing Nus tag and S tag) was highly expressed in E. coli. The total protein was about 80%. The fusion protein was successfully excised and the unlabeled fusion protein was purified. The relative molecular mass of the fusion protein was about 17 000 by Western blotting. The results of gel permeation retardation assay showed that the fusion protein 3TAT-DRBD could effectively bind to the target survivin siRNA (survivin-siRNA). Confocal laser scanning microscopy showed that the efficiency of survivin-siRNA penetrating the cell membrane into prostate cancer PC3 cells was significantly increased under TAT-mediated transfection. CONCLUSION: The 3TAT-DRBD fusion protein with siRNA binding activity and transmembrane function was successfully expressed and purified, which laid the foundation for functional studies and clinical application of 3TAT-DRBD.