本文關(guān)鍵詞： IL-31 高效表達(dá) 最佳條件 皮膚炎癥　出處：《遵義醫(yī)學(xué)院》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:探索最佳的重組人IL-31的高效表達(dá)條件,獲得高純度的重組蛋白,并用獲得的rhIL-31致小鼠皮膚炎癥,探討rhIL-31與皮膚炎癥的直接相關(guān)性及其作用機(jī)制。方法:(1)培養(yǎng)工程菌株E.coli BL21(DE3),SDS-PAGE分析不同誘導(dǎo)劑濃度、不同誘導(dǎo)溫度、不同誘導(dǎo)時(shí)間的條件下重組蛋白表達(dá)量,探索適宜的表達(dá)條件。(2)溶菌酶結(jié)合超聲波處理破碎細(xì)菌,去垢劑洗滌雜蛋白,溶解包涵體,用sephadexG-75和Ni-NTA瓊脂糖凝膠FF柱分別純化rhIL-31溶包液,SDS-PAGE分析純化效果,Western blot、氨基酸序列分析鑒定rhIL-31。(3)純化產(chǎn)物進(jìn)行透析濃縮,測(cè)定濃縮后蛋白濃度。(4)用獲得的復(fù)性后rhIL-31蛋白以10μg、20μg、40μg三種不同劑量對(duì)Balb/C小鼠進(jìn)行皮下注射和肌肉注射,并設(shè)相應(yīng)途徑對(duì)照組,觀察其皮膚和行為變化,體重和外周血白細(xì)胞變化;注射10天后取皮損部位皮膚作石蠟切片并進(jìn)行HE染色,觀察皮膚組織的炎性細(xì)胞浸潤(rùn)情況,同時(shí)收集血清,檢測(cè)小鼠血清中IL-6,IL-8,MIP-3β和L選擇素等炎性細(xì)胞因子水平。結(jié)果:(1)SDS-PAGE分析發(fā)現(xiàn),IPTG濃度為1.0mmol/L,溫度37℃左右,誘導(dǎo)5h蛋白表達(dá)量最高。(2)sephadexG-75目的蛋白和雜蛋白同時(shí)洗脫;Ni-NTA瓊脂糖凝膠FF柱純化蛋白包涵體溶解液50mM咪唑洗脫雜蛋白,100mM、200mM咪唑洗脫目的蛋白。(3)Western blot可見目的蛋白約34kd處有特異性蛋白條帶;氨基酸序列分析證實(shí),我們獲得的重組蛋白與人IL-31氨基酸序列一致。(4)注射rhIL-31的小鼠出現(xiàn)頻繁的搔抓行為,有脫毛現(xiàn)象,小鼠體重增幅降低;外周血白細(xì)胞總數(shù)增加,中性粒細(xì)胞顯著增高,有一定的劑量依賴性。(5)小鼠皮膚病理HE染色可見大量炎性細(xì)胞浸潤(rùn),深達(dá)肌層,受損皮膚組織出現(xiàn)棘層肥厚,高劑量實(shí)驗(yàn)組可見肌肉溶解。(6)兩種注射途徑與其對(duì)照組比較,小鼠血清細(xì)胞因子IL-6、IL-8、L-選擇素和MIP-3β水平均有顯著增高(p＜0.05),并且隨IL-31劑量的增加而增高,具有劑量依賴性;皮下注射實(shí)驗(yàn)組IL-6、L-選擇素和MIP-3B水平略低于肌肉注射實(shí)驗(yàn)組,IL-8水平接近。結(jié)論:(1)IPTG濃度為1.0mmol/L,溫度37℃左右,誘導(dǎo)5h蛋白表達(dá)量最高。(2)用和Ni-NTA瓊脂糖凝膠FF柱純化rhIL-31效果優(yōu)于sephadex G-75。(3)注射rhIL-31的小鼠體重增幅降低,有頻繁的搔抓行為,可見脫毛;外周血白細(xì)胞總數(shù)增加,中性粒細(xì)胞數(shù)增加;皮膚病理HE染色見棘層肥厚,大量炎性細(xì)胞浸潤(rùn),高劑量實(shí)驗(yàn)組出現(xiàn)肌肉溶解,證明rhIL-31能夠誘發(fā)小鼠皮膚炎癥。(4)小鼠血清細(xì)胞因子IL-6、IL-8、L-選擇素和MIP-3β水平隨rhIL-31劑量的增加而增高,且肌肉注射組高于皮下注射組,表明rhIL-31可能通過(guò)誘導(dǎo)表達(dá)趨化因子和炎性因子發(fā)揮其致炎作用。
[Abstract]:Objective: to explore the best expression conditions of recombinant human IL-31, to obtain the recombinant protein with high purity and to induce skin inflammation in mice with the obtained rhIL-31. Objective: to investigate the direct relationship between rhIL-31 and skin inflammation and its mechanism. Methods the engineering strain E.coli BL21DE3 was cultured by 1: 1. SDS-PAGE was used to analyze the expression of recombinant protein under different inducer concentration, different induction temperature and different induction time. To explore the appropriate expression conditions. 2) lysozyme combined with ultrasonic treatment of broken bacteria, detergent washing miscellaneous protein, soluble inclusion body. SephadexG-75 and Ni-NTA agarose gel FF column were used to purify rhIL-31 dissolution solution respectively. SDS-PAGE was used to analyze the purification effect. Western blot, amino acid sequence analysis and identification of rhIL-31. 3) purified products for dialysis concentration. The concentration of concentrated protein was determined. The rhIL-31 protein was refolded at 10 渭 g or 20 渭 g. Three different doses of 40 渭 g were injected subcutaneously and intramuscularly into Balb/C mice, and the corresponding control group was set up to observe the changes of skin and behavior, body weight and peripheral white blood cells. After 10 days of injection, paraffin sections were taken from the skin lesions and stained with HE to observe the infiltration of inflammatory cells in the skin tissue. At the same time, the serum was collected, and the IL-6 IL-8 in the serum of mice was detected. Results the concentration of MIP-3 尾 and L-selectin was 1.0 mmol / L and the temperature was about 37 鈩，

本文編號(hào)：1446609