【摘要】：目的：構(gòu)建pGEX-6p-1/porB原核重組質(zhì)粒，表達(dá)并純化GST-PorB融合蛋白，分析重組PorB蛋白誘導(dǎo)人單核細(xì)胞白血病細(xì)胞（THP-1）分泌促炎細(xì)胞因子IL-1β、TNF-α及HMGB1的能力，并探討PorB蛋白對(duì)誘導(dǎo)THP-1細(xì)胞凋亡作用的影響。 方法：從Genbank獲得PorB蛋白的全基因序列，設(shè)計(jì)引物，使用PCR技術(shù)擴(kuò)增出PorB蛋白的基因序列，通過(guò)基因克隆技術(shù)將該段序列插入到pGEX-6p-1中，構(gòu)建pGEX-6p-1/porB原核表達(dá)載體，以雙酶切及測(cè)序鑒定載體構(gòu)建是否成功。將構(gòu)建好的載體轉(zhuǎn)入到制備好的Ecoli-BL-21感受態(tài)細(xì)菌中，誘導(dǎo)表達(dá)PorB蛋白，摸索最適誘導(dǎo)表達(dá)條件；通過(guò)尿素濃度梯度復(fù)性法對(duì)包涵體PorB蛋白進(jìn)行復(fù)性，使用GST-Bind樹脂純化復(fù)性后的PorB蛋白。BCA法測(cè)定純化后的蛋白濃度，使用多粘菌素-B去除蛋白中的內(nèi)毒素，鱟試劑盒測(cè)定內(nèi)毒素含量。PorB蛋白經(jīng)過(guò)處理后分別以時(shí)間梯度和濃度梯度作用于THP-1細(xì)胞， ELISA雙抗體夾心法分別檢測(cè)IL-1β、TNF-α和HMGB1的產(chǎn)生水平；采用流式細(xì)胞術(shù)和AnnexinV-EGFP-PI雙染色法觀察不同時(shí)間和不同濃度的PorB蛋白對(duì)誘導(dǎo)THP-1細(xì)胞凋亡作用的影響。 結(jié)果： (1)正確克隆porB基因，并將porB基因插入到pGEX-6p-1載體中，成功構(gòu)建了porB/pGEX-6p-1原核表達(dá)載體。 (2)摸索出重組蛋白的最適表達(dá)條件，即在30℃時(shí)，以0.2mM IPTG誘導(dǎo)表達(dá)菌表達(dá)4h時(shí)，蛋白表達(dá)效果最好。經(jīng)純化后的重組蛋白，通過(guò)SDS-PAGE電泳，只出現(xiàn)大小為59KD左右的單一條帶，符合GST-PorB融合蛋白的預(yù)測(cè)值。 (3)分別以濃度為1，3，5，，7，9μg/ml的重組porB蛋白處理THP-1細(xì)胞24h，在蛋白濃度為5μg/ml時(shí)，TNF-α，IL-1β和HMGB1分泌量達(dá)到最高峰，分別為（27.10±1.23）pg/ml，（109.44±3.53）pg/ml，（320.09±9.67）pg/ml，與PBS/GST對(duì)照組及其它各濃度組比較具有顯著差異(P0.05)。使用5μg/ml濃度的重組蛋白分別刺激THP-1細(xì)胞6，12，24，36，48h，TNF-α，IL-1β在刺激時(shí)間為36h時(shí)分泌量最高，分別為（39.94±2.35）pg/ml，（223.14±7.27）pg/ml，與其它各時(shí)間點(diǎn)組比較具有顯著差異(P0.05)，而HMGB1分泌量從6h至48小時(shí)之間一直呈上升趨勢(shì)。 (4)分別以濃度為1，3，5，7，9μg/ml的重組porB蛋白濃度處理THP-1細(xì)胞24h，通過(guò)AnnexinV-EGFP-PI熒光雙染法和流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡情況，在蛋白濃度為7μg/ml時(shí)，細(xì)胞凋亡情況最明顯，凋亡百分率為（23.13±2.01）％，與PBS/GST對(duì)照組及其它各濃度組比較具有顯著差異(P0.05)；使用7μg/ml的蛋白處理細(xì)胞0，12，24，36，48h，結(jié)果顯示在24h時(shí)細(xì)胞凋亡率達(dá)到最高，為（20.75±2.03）％，與其它各時(shí)間點(diǎn)組比較具有顯著差異(P0.05)。 結(jié)論： (1) PorB重組蛋白能通過(guò)時(shí)間和劑量依賴方式誘導(dǎo)THP-1細(xì)胞分泌較高水平的促炎細(xì)胞因子IL-1β、TNF-α和HMGB1。 (2) PorB重組蛋白能通過(guò)時(shí)間和劑量依賴方式誘導(dǎo)THP-1細(xì)胞發(fā)生凋亡。
[Abstract]:Objective: to construct the prokaryotic recombinant plasmid of pGEX-6p-1/porB, express and purify GST-PorB fusion protein, and analyze the ability of recombinant PorB protein to induce the secretion of pro-inflammatory cytokines IL-1 尾, TNF- 偽 and HMGB1 by human monocytic leukemia cells (THP-1). The effect of PorB protein on apoptosis of THP-1 cells was also investigated. Methods: the whole gene sequence of PorB protein was obtained from Genbank, the primers were designed, the gene sequence of PorB protein was amplified by PCR, and inserted into pGEX-6p-1 by gene cloning technology. The prokaryotic expression vector of pGEX-6p-1/porB was constructed, and the success of the vector was identified by double enzyme digestion and sequencing. The constructed vector was transferred into the prepared Ecoli-BL-21 competent bacteria to induce the expression of PorB protein and to explore the optimal conditions for inducing the expression. The inclusion body PorB protein was renatured by urea concentration gradient renaturation, the renatured PorB protein was purified by GST-Bind resin, the purified protein concentration was determined by BCA method, and the endotoxin in the protein was removed by polymyxin-B. THP-1 cells were treated with time gradient and concentration gradient, respectively. IL-1 尾, TNF- 偽 and HMGB1 production levels were detected by ELISA double antibody sandwich method. The effects of different time and concentration of PorB protein on apoptosis of THP-1 cells were observed by flow cytometry and AnnexinV-EGFP-PI double staining. Results: (1) the porB gene was cloned correctly, and the porB gene was inserted into the pGEX-6p-1 vector. The prokaryotic expression vector of porB/pGEX-6p-1 was constructed successfully. (2) the optimal expression condition of the recombinant protein was found, that is, at 30 鈩

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