核转运抑制肽表达载体的构建及对HeLa细胞增殖、迁移的影响

发布时间：2018-01-18 00:16

本文关键词：核转运抑制肽表达载体的构建及对HeLa细胞增殖、迁移的影响　出处：《四川大学学报(医学版)》2017年06期 　论文类型：期刊论文

【摘要】：目的构建核转运抑制肽与红色荧光蛋白(RFP)的融合表达载体,探讨该融合蛋白在HeLa细胞中的表达、定位以及对细胞增殖、迁移的影响。方法分别合成编码两种核转运抑制肽Bimax1和Bimax2的多核苷酸,退火后插入红色荧光蛋白表达载体pDs-Red-C1;质粒转化大肠杆菌DH-5α,挑取阳性菌进行DNA测序;利用脂质体转染法将重组载体pDs-Red-Bimax1、pDs-Red-Bimax2及空白载体转入HeLa细胞中,荧光显微镜观察红色荧光的表达与定位;MTT实验和细胞迁移实验检测融合蛋白表达对细胞增殖、迁移的影响。结果 DNA测序显示,两种核转运抑制肽Bimax1和Bimax2成功插入红色荧光蛋白表达载体;转染真核细胞后Bimax1和Bimax2引导红色荧光蛋白在细胞核表达;同时,融合蛋白RFP-Bimax1和RFP-Bimax2对HeLa细胞的增殖、迁移有抑制作用。结论成功构建核转运特异性抑制肽与红色荧光蛋白融合表达的载体,融合蛋白在细胞核局限表达并能抑制肿瘤细胞的增殖、迁移。
[Abstract]:Objective to construct a fusion expression vector of nuclear transport inhibitory peptide (NPP) and red fluorescent protein (RFP), and to investigate the expression, localization and proliferation of the fusion protein in HeLa cells. Methods the polynucleotides encoding two nuclear transport inhibitory peptides (Bimax1 and Bimax2) were synthesized and inserted into the red fluorescent protein expression vector pDs-Red-C1 after annealing. The plasmid was transformed into Escherichia coli DH-5 伪, and the positive bacteria were selected for DNA sequencing. The recombinant vector pDs-Red-Bimax2 and the blank vector pDs-Red-Bimax2 were transfected into HeLa cells by liposome transfection. The expression and localization of red fluorescence were observed by fluorescence microscope. MTT assay and cell migration assay were used to detect the effect of fusion protein expression on cell proliferation and migration. Results DNA sequencing showed. Two nuclear transport inhibitory peptides, Bimax1 and Bimax2, were successfully inserted into the red fluorescent protein expression vector. After transfection of eukaryotic cells, Bimax1 and Bimax2 guided the expression of red fluorescent protein in the nucleus. At the same time, the proliferation of HeLa cells was induced by the fusion proteins RFP-Bimax1 and RFP-Bimax2. Conclusion the fusion expression vector of nuclear transporter specific inhibitory peptide and red fluorescent protein is successfully constructed. The fusion protein is localized expressed in the nucleus and can inhibit the proliferation and migration of tumor cells.
【作者单位】：江西省系统生物医学重点实验室;九江学院临床医学院技能中心;
【基金】：国家自然科学基金(No.81360143) 江西省自然科学基金(No.20151BAB205056)资助
【分类号】：R3416
【正文快照】： 细胞核与细胞质的区间分布是真核细胞的显著标志,胞质中翻译的核蛋白跨越核膜在细胞核与细胞质之间的穿梭受到严格调节。大量研究表明,多种疾病包括肿瘤、糖尿病及中枢退行性疾病等的形成与核蛋白的转运异常有关[1-2]。因此,探明关键蛋白质的入核转运机制和调节因素,对于研究

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