【摘要】：目的:構(gòu)建rh-S100A9原核表達質(zhì)粒pET 28a-S100A9,誘導表達、分離、純化上清和包涵體來源的rh-S100A9蛋白。比較兩種來源的S100A9蛋白對神經(jīng)母細胞瘤細胞株SH-SY5Y的生物學作用,并初步探索該蛋白對SH-SY5Y細胞生物學效應的相關機制。方法:運用全基因合成法合成人S100A9基因,構(gòu)建重組人S100A9原核表達質(zhì)粒pET28a-S100A9,經(jīng)雙酶切法及聚合酶鏈式反應(Polymerase Chain Reaction,PCR)法對重組質(zhì)粒進行鑒定。在不同條件下,使用異丙基硫代β-D-半乳糖苷(IPTG)分別誘導rh-S100A9蛋白表達,經(jīng)聚丙烯酰胺凝膠電泳(SDS-PAGE)和Western blot進行鑒定。采用鎳親和柱分離并純化兩種來源重組蛋白,透析并低溫凍干獲得S100A9蛋白粉。采用CCK-8法檢測上清和包涵體來源的S100A9蛋白對SH-SY5Y細胞活性的影響。采用AO/EB雙重染色,流式細胞術(shù)檢測細胞周期、凋亡,DCFH-DA探針法檢測細胞活性氧等方法初步探討S100A9對SH-SY5Y細胞的增殖抑制的作用機理。結(jié)果:成功構(gòu)建了rh-S100A9原核表達質(zhì)粒pET 28a-S100A9;在18℃誘導表達時,獲得大量表達于上清中的S100A9蛋白,而在37℃時S100A9大量表達于包涵體中。在濃度為0.05 mg/mL時,上清和包涵體來源的rh-S100A9對SH-SY5Y具有明顯增殖抑制作用(P0.01),二者作用無顯著性差異。AO/EB染色及流式細胞術(shù)結(jié)果表明兩種來源S100A9均可誘導SH-SY5Y細胞發(fā)生凋亡,經(jīng)統(tǒng)計分析顯示二者對于SH-SY5Y細胞作用無顯著性差異。細胞周期檢測結(jié)果顯示S100A9可將細胞周期阻滯在G2/M期。ROS檢測結(jié)果表明S100A9可激活SH-SY5Y細胞內(nèi)活性氧的產(chǎn)生。結(jié)論:本研究成功獲取足量兩種來源rh-S100A9重組蛋白,且兩種來源S100A9蛋白作用于SH-SY5Y細胞所產(chǎn)生增殖抑制生物學效應一致。S100A9可通過促進SH-SY5Y細胞的凋亡抑制其增殖,并能顯著將細胞周期阻滯在G2/M期。S100A9可促進細胞內(nèi)ROS產(chǎn)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，

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