本文選題：親和純化 + SA　； 參考：《中南大學(xué)學(xué)報(醫(yī)學(xué)版)》2017年04期

【摘要】：目的:利用親和純化技術(shù)聯(lián)合質(zhì)譜分析篩選鑒定與S100A8和S100A9相互作用的蛋白,為探討S100A8和S100A9在結(jié)腸炎相關(guān)性結(jié)腸癌發(fā)生發(fā)展中的作用機制提供線索。方法:構(gòu)建含有S100A8和S100A9 CDS區(qū)和Flag親和標(biāo)簽的真核表達載體p3×Flag-CMV-S100A8和p3×Flag-CMV-S100A9,將其轉(zhuǎn)染至HEK293細胞中,48 h后收集蛋白。運用標(biāo)簽分離純化S100A8和S100A9蛋白,液相質(zhì)譜/質(zhì)譜分析技術(shù)(liquid chromatography mass spectrometry/mass spectrometry,LC-MS/MS)鑒定S100A8和S100A9的相互作用蛋白,通過生物信息學(xué)的方法(GO分析)歸納分析相互作用蛋白,免疫共沉淀實驗鑒定S100A8和S100A9的相互作用蛋白。結(jié)果:通過標(biāo)簽純化聯(lián)合質(zhì)譜分析成功鑒定出PKM,NPM1,e IF5A等14個與S100A8相互作用的蛋白,鑒定出14-3-3ε,PKM等6個與S100A9相互作用的蛋白。對所鑒定的相互作用蛋白進行生物信息學(xué)分析,發(fā)現(xiàn)與S100A8和S100A9相互作用的蛋白參與糖代謝、細胞黏附、正向調(diào)控NF-κB轉(zhuǎn)錄因子活性、抗凋亡等在內(nèi)的生物信號通路,并利用免疫共沉淀證實PKM2與S100A8和S100A9發(fā)生相互作用,14-3-3ε與S100A9發(fā)生相互作用。結(jié)論:利用免疫共沉淀結(jié)合質(zhì)譜技術(shù)篩選鑒定的S100A8和S100A9相互作用蛋白PKM2和14-3-3ε,可能為闡明S100A8和S100A9在結(jié)腸炎相關(guān)性結(jié)腸癌發(fā)生發(fā)展中的作用機制提供重要線索。
[Abstract]:Objective: to screen and identify proteins interacting with S100A8 and S100A9 by affinity purification and mass spectrometry in order to provide clues to the role of S100A8 and S100A9 in the development of colitis associated colon cancer. Methods: eukaryotic expression vectors p3 脳 Flag-CMV-S100A8 and p3 脳 Flag-CMV-S100A9 containing S100A8 and S100A9 CDS regions and Flag affinity tags were constructed and transfected into HEK293 cells for 48 h to collect proteins. S100A8 and S100A9 proteins were separated and purified by label, and the interaction proteins between S100A8 and S100A9 were identified by liquid phase mass spectrometry / mass spectrometry (LC-MS / MS). The interaction proteins were analyzed by bioinformatics method (go analysis). The interaction protein between S100A8 and S100A9 was identified by immunoprecipitation assay. Results: 14 proteins interacting with S100A8 were successfully identified by tag purification and mass spectrometry, and 6 proteins interacting with S100A9 were identified, such as 14-3-3 蔚 -PKM. Bioinformatics analysis of the identified interacting proteins showed that the proteins interacting with S100A8 and S100A9 were involved in glucose metabolism, cell adhesion, positive regulation of NF- 魏 B transcription factor activity, anti-apoptosis, and so on. The interaction of PKM2 with S100A8 and S100A9 was confirmed by immunoprecipitation. 14-3-3 蔚 interacted with S100A9. Conclusion: the identification of PKM2 and 14-3-3 蔚 of S100A8 and S100A9 interaction proteins by immunoprecipitation and mass spectrometry may provide important clues for elucidating the role of S100A8 and S100A9 in the pathogenesis of colitis associated colon cancer.
【作者單位】： 中南大學(xué)湘雅三醫(yī)院消化內(nèi)科;中南大學(xué)湘雅三醫(yī)院湖南省非可控性炎癥與腫瘤重點實驗室;中南大學(xué)腫瘤研究所;中南大學(xué)高等研究中心高分辨質(zhì)譜實驗室;
【基金】：國家自然科學(xué)基金(81472286) 湖南省自然科學(xué)基金(2016JJ6153) 中南大學(xué)博士生自主探索創(chuàng)新項目(2015zzts100);中南大學(xué)米塔爾學(xué)生創(chuàng)新項目(15MX47) 沈守榮教授的湘雅名醫(yī)基金~~
【分類號】：R3411
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本文編號：1842292