本文選題：NF-κB + 熒光素酶報(bào)告基因　； 參考：《廣西醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的: NF-κB是普遍存在于細(xì)胞漿中的一種轉(zhuǎn)錄調(diào)節(jié)因子,很多疾病與NF-κB信號(hào)通路的激活和抑制有關(guān)。本課題建立了適合大量微生物代謝產(chǎn)物提取物的篩選和提取方法，采用雙熒光素酶報(bào)告基因分析方法研究525種代謝產(chǎn)物對(duì)NF-κB信號(hào)通路的激活和抑制情況，對(duì)篩選得到的活性菌株進(jìn)行發(fā)酵罐發(fā)酵，對(duì)分離得到的化合物進(jìn)行結(jié)構(gòu)鑒定和生物活性評(píng)價(jià)，然后進(jìn)一步做相關(guān)基礎(chǔ)研究。 方法：微生物接種液體培養(yǎng)基中，于180r/min、30℃搖床培養(yǎng)，3d后，加入終濃度70%的乙醇裂解過夜。裂解物離心，取上清，旋轉(zhuǎn)蒸發(fā)濃縮并冷凍干燥，獲得微生物代謝產(chǎn)物提取物，采用雙熒光素酶報(bào)告基因分析方法進(jìn)行篩選，將pNF-ΚB-luc、pRL-TK質(zhì)粒共轉(zhuǎn)染HEK293細(xì)胞，轉(zhuǎn)染后6h加入微生物代謝產(chǎn)物或TNFα（陽(yáng)性對(duì)照），繼續(xù)培養(yǎng)24h后，檢測(cè)熒光素酶活性。 對(duì)3092#活性菌株進(jìn)行大規(guī)模發(fā)酵，培養(yǎng)條件為可溶性淀粉1%、葡萄糖2%、牛肉膏0.1%、NaCl0.2%、K2HPO40.025%、酵母提取物1%、CaCO30.2%，PH=7。我們以此條件進(jìn)行了三批發(fā)酵罐發(fā)酵，，發(fā)酵液經(jīng)過初步提取后得到35.7g粗提物。粗提物采用硅膠柱色譜、凝膠柱色譜、TLC、HPLC等分離方法進(jìn)行分離，化合物采用HR-MS、13C-NMR、1H-NMR等方法鑒別。 對(duì)分離到的單體化合物進(jìn)行雙熒光素酶報(bào)告基因分析，研究這些化合物對(duì)NF-κB信號(hào)通路的激活和抑制情況 結(jié)果：在第一輪篩選中，發(fā)現(xiàn)30種代謝物能夠激活NF-κB信號(hào)通路；28種代謝物能夠抑制NF-κB信號(hào)通路。在第二輪篩選中，有13種代謝物能夠明顯激活NF-κB信號(hào)通路，沒有明顯抑制NF-κB信號(hào)通路的代謝物。在第三輪篩選和最后驗(yàn)證中，發(fā)現(xiàn)６種代謝物均能激活NF-κB信號(hào)通路。 從粗提物中得到一系列脂肽地衣素類物質(zhì)（L31-L37）,通過與文獻(xiàn)比對(duì)，確定結(jié)構(gòu)如下： L32：cyclo-[L-Gln1→L-Leu2→D-Leu3→L-Val4→L-Asp5→D-Leu6→L-Ile7]-β-OH fatty acid (isoC13)和cyclo-[L-Gln1→L-Leu2→D-Leu3→L-Val4→L-Asp5→D-Leu6→L-Ile7]-β-OH fatty acid (anteisoC13) L34：cyclo-[L-Gln1→L-Leu2→D-Leu3→L-Val4→L-Asp5→D-Leu6→L-Ile7]-β-OH fatty acid (isoC14)。 L36：cyclo-[L-Gln1→L-Leu2→D-Leu3→L-Val4→L-Asp5→D-Leu6→L-Ile7]-β-OH fatty acid (isoC15)和cyclo-[L-Gln1→L-Leu2→D-Leu3→L-Val4→L-Asp5→D-Leu6→L-Ile7]-β-OH fatty acid (anteisoC15) L33：cyclo-[L-Gln1→L-Leu2→D-Leu3→L-Val4→L-Asp5→D-Leu6→L-Ile7]-β-OH fatty acid (isoC13或anteisoC13)和cyclo-[L-Gln1→L-Leu2→D-Leu3→L-Val4→L-Asp5→D-Leu6→L-Val7]-β-OH fatty acid(isoC14或anteisoC14) L35：cyclo-[L-Gln1→L-Leu2→D-Leu3→L-Val4→L-Asp5→D-Leu6→L-Ile7]-β-OH fatty acid (nC14)和cyclo-[L-Gln1→L-Leu2→D-Leu3→L-Val4→L-Asp5→D-Leu6→L-Val7]-β-OH fatty acid (nC15) 此外，還分離到5個(gè)其他類單體化合物，分離鑒定了其中三個(gè)，名稱分別為亮氨酸、正十六烷酸、3-羥基癸烷酸。
[Abstract]:Objective: NF- 魏 B is a transcriptional regulator in cytoplasm. Many diseases are related to the activation and inhibition of NF- 魏 B signaling pathway. In this study, a suitable method for screening and extracting a large number of microbial metabolite extracts was established. The activation and inhibition of 525 metabolites on NF- 魏 B signaling pathway were studied by double luciferase reporter gene analysis. The active strains were fermented in fermenter, the compounds were identified by structure identification and bioactivity evaluation, and further basic research was done. Methods: microbes were inoculated in liquid medium and incubated in a shaking bed at 180 r / min ~ 30 鈩

本文編號(hào)：1956506