rh-S100A9质粒构建及蛋白表达、分离纯化和其生物学效应
发布时间：2018-12-31 14:00
【摘要】：目的:构建rh-S100A9原核表达质粒pET 28a-S100A9,诱导表达、分离、纯化上清和包涵体来源的rh-S100A9蛋白。比较两种来源的S100A9蛋白对神经母细胞瘤细胞株SH-SY5Y的生物学作用,并初步探索该蛋白对SH-SY5Y细胞生物学效应的相关机制。方法:运用全基因合成法合成人S100A9基因,构建重组人S100A9原核表达质粒pET28a-S100A9,经双酶切法及聚合酶链式反应(Polymerase Chain Reaction,PCR)法对重组质粒进行鉴定。在不同条件下,使用异丙基硫代β-D-半乳糖苷(IPTG)分别诱导rh-S100A9蛋白表达,经聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot进行鉴定。采用镍亲和柱分离并纯化两种来源重组蛋白,透析并低温冻干获得S100A9蛋白粉。采用CCK-8法检测上清和包涵体来源的S100A9蛋白对SH-SY5Y细胞活性的影响。采用AO/EB双重染色,流式细胞术检测细胞周期、凋亡,DCFH-DA探针法检测细胞活性氧等方法初步探讨S100A9对SH-SY5Y细胞的增殖抑制的作用机理。结果:成功构建了rh-S100A9原核表达质粒pET 28a-S100A9;在18℃诱导表达时,获得大量表达于上清中的S100A9蛋白,而在37℃时S100A9大量表达于包涵体中。在浓度为0.05 mg/mL时,上清和包涵体来源的rh-S100A9对SH-SY5Y具有明显增殖抑制作用(P0.01),二者作用无显著性差异。AO/EB染色及流式细胞术结果表明两种来源S100A9均可诱导SH-SY5Y细胞发生凋亡,经统计分析显示二者对于SH-SY5Y细胞作用无显著性差异。细胞周期检测结果显示S100A9可将细胞周期阻滞在G2/M期。ROS检测结果表明S100A9可激活SH-SY5Y细胞内活性氧的产生。结论:本研究成功获取足量两种来源rh-S100A9重组蛋白,且两种来源S100A9蛋白作用于SH-SY5Y细胞所产生增殖抑制生物学效应一致。S100A9可通过促进SH-SY5Y细胞的凋亡抑制其增殖,并能显著将细胞周期阻滞在G2/M期。S100A9可促进细胞内ROS产生增加。
[Abstract]:Aim: to construct rh-S100A9 prokaryotic expression plasmid pET 28a-S100A9 and express, isolate and purify rh-S100A9 protein from supernatant and inclusion body. To compare the biological effects of two S100A9 proteins on neuroblastoma cell line SH-SY5Y, and to explore the mechanism of the biological effects of the protein on SH-SY5Y cells. Methods: the human S100A9 gene was synthesized by whole gene synthesis method, and the recombinant human S100A9 prokaryotic expression plasmid pET28a-S100A9, was identified by double enzyme digestion and polymerase chain reaction (Polymerase Chain Reaction,PCR). The expression of rh-S100A9 protein was induced by isopropyl thiothioside (IPTG) under different conditions and was identified by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Two recombinant proteins were isolated and purified by nickel affinity column. S100A9 protein powder was obtained by dialysis and freeze-drying at low temperature. The effect of S100A9 protein from supernatant and inclusion body on the activity of SH-SY5Y cells was detected by CCK-8 assay. Cell cycle, apoptosis and reactive oxygen species (Ros) were detected by AO/EB double staining, flow cytometry and DCFH-DA probe method respectively. The mechanism of S100A9 inhibiting proliferation of SH-SY5Y cells was studied. Results: rh-S100A9 prokaryotic expression plasmid pET 28a-S100A9 was successfully constructed, and a large number of S100A9 protein expressed in supernatant was obtained at 18 鈩，
本文编号：2396678
[Abstract]:Aim: to construct rh-S100A9 prokaryotic expression plasmid pET 28a-S100A9 and express, isolate and purify rh-S100A9 protein from supernatant and inclusion body. To compare the biological effects of two S100A9 proteins on neuroblastoma cell line SH-SY5Y, and to explore the mechanism of the biological effects of the protein on SH-SY5Y cells. Methods: the human S100A9 gene was synthesized by whole gene synthesis method, and the recombinant human S100A9 prokaryotic expression plasmid pET28a-S100A9, was identified by double enzyme digestion and polymerase chain reaction (Polymerase Chain Reaction,PCR). The expression of rh-S100A9 protein was induced by isopropyl thiothioside (IPTG) under different conditions and was identified by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Two recombinant proteins were isolated and purified by nickel affinity column. S100A9 protein powder was obtained by dialysis and freeze-drying at low temperature. The effect of S100A9 protein from supernatant and inclusion body on the activity of SH-SY5Y cells was detected by CCK-8 assay. Cell cycle, apoptosis and reactive oxygen species (Ros) were detected by AO/EB double staining, flow cytometry and DCFH-DA probe method respectively. The mechanism of S100A9 inhibiting proliferation of SH-SY5Y cells was studied. Results: rh-S100A9 prokaryotic expression plasmid pET 28a-S100A9 was successfully constructed, and a large number of S100A9 protein expressed in supernatant was obtained at 18 鈩，
本文编号：2396678
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