本文關(guān)鍵詞：核轉(zhuǎn)運(yùn)抑制肽表達(dá)載體的構(gòu)建及對HeLa細(xì)胞增殖、遷移的影響　出處：《四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2017年06期 　論文類型：期刊論文

  更多相關(guān)文章： 核轉(zhuǎn)運(yùn) 紅色熒光蛋白 增殖 遷移

【摘要】：目的構(gòu)建核轉(zhuǎn)運(yùn)抑制肽與紅色熒光蛋白(RFP)的融合表達(dá)載體,探討該融合蛋白在HeLa細(xì)胞中的表達(dá)、定位以及對細(xì)胞增殖、遷移的影響。方法分別合成編碼兩種核轉(zhuǎn)運(yùn)抑制肽Bimax1和Bimax2的多核苷酸,退火后插入紅色熒光蛋白表達(dá)載體pDs-Red-C1;質(zhì)粒轉(zhuǎn)化大腸桿菌DH-5α,挑取陽性菌進(jìn)行DNA測序;利用脂質(zhì)體轉(zhuǎn)染法將重組載體pDs-Red-Bimax1、pDs-Red-Bimax2及空白載體轉(zhuǎn)入HeLa細(xì)胞中,熒光顯微鏡觀察紅色熒光的表達(dá)與定位;MTT實(shí)驗(yàn)和細(xì)胞遷移實(shí)驗(yàn)檢測融合蛋白表達(dá)對細(xì)胞增殖、遷移的影響。結(jié)果 DNA測序顯示,兩種核轉(zhuǎn)運(yùn)抑制肽Bimax1和Bimax2成功插入紅色熒光蛋白表達(dá)載體;轉(zhuǎn)染真核細(xì)胞后Bimax1和Bimax2引導(dǎo)紅色熒光蛋白在細(xì)胞核表達(dá);同時(shí),融合蛋白RFP-Bimax1和RFP-Bimax2對HeLa細(xì)胞的增殖、遷移有抑制作用。結(jié)論成功構(gòu)建核轉(zhuǎn)運(yùn)特異性抑制肽與紅色熒光蛋白融合表達(dá)的載體,融合蛋白在細(xì)胞核局限表達(dá)并能抑制腫瘤細(xì)胞的增殖、遷移。
[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 偽, 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.
【作者單位】： 江西省系統(tǒng)生物醫(yī)學(xué)重點(diǎn)實(shí)驗(yàn)室;九江學(xué)院臨床醫(yī)學(xué)院技能中心;
【基金】：國家自然科學(xué)基金(No.81360143) 江西省自然科學(xué)基金(No.20151BAB205056)資助
【分類號】：R3416
【正文快照】： 細(xì)胞核與細(xì)胞質(zhì)的區(qū)間分布是真核細(xì)胞的顯著標(biāo)志,胞質(zhì)中翻譯的核蛋白跨越核膜在細(xì)胞核與細(xì)胞質(zhì)之間的穿梭受到嚴(yán)格調(diào)節(jié)。大量研究表明,多種疾病包括腫瘤、糖尿病及中樞退行性疾病等的形成與核蛋白的轉(zhuǎn)運(yùn)異常有關(guān)[1-2]。因此,探明關(guān)鍵蛋白質(zhì)的入核轉(zhuǎn)運(yùn)機(jī)制和調(diào)節(jié)因素,對于研究

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