重组人IL-31高效表达、纯化及致小鼠皮肤炎症的研究

发布时间：2018-01-20 03:13

  本文关键词： IL-31 高效表达 最佳条件 皮肤炎症　出处：《遵义医学院》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:探索最佳的重组人IL-31的高效表达条件,获得高纯度的重组蛋白,并用获得的rhIL-31致小鼠皮肤炎症,探讨rhIL-31与皮肤炎症的直接相关性及其作用机制。方法:(1)培养工程菌株E.coli BL21(DE3),SDS-PAGE分析不同诱导剂浓度、不同诱导温度、不同诱导时间的条件下重组蛋白表达量,探索适宜的表达条件。(2)溶菌酶结合超声波处理破碎细菌,去垢剂洗涤杂蛋白,溶解包涵体,用sephadexG-75和Ni-NTA琼脂糖凝胶FF柱分别纯化rhIL-31溶包液,SDS-PAGE分析纯化效果,Western blot、氨基酸序列分析鉴定rhIL-31。(3)纯化产物进行透析浓缩,测定浓缩后蛋白浓度。(4)用获得的复性后rhIL-31蛋白以10μg、20μg、40μg三种不同剂量对Balb/C小鼠进行皮下注射和肌肉注射,并设相应途径对照组,观察其皮肤和行为变化,体重和外周血白细胞变化;注射10天后取皮损部位皮肤作石蜡切片并进行HE染色,观察皮肤组织的炎性细胞浸润情况,同时收集血清,检测小鼠血清中IL-6,IL-8,MIP-3β和L选择素等炎性细胞因子水平。结果:(1)SDS-PAGE分析发现,IPTG浓度为1.0mmol/L,温度37℃左右,诱导5h蛋白表达量最高。(2)sephadexG-75目的蛋白和杂蛋白同时洗脱;Ni-NTA琼脂糖凝胶FF柱纯化蛋白包涵体溶解液50mM咪唑洗脱杂蛋白,100mM、200mM咪唑洗脱目的蛋白。(3)Western blot可见目的蛋白约34kd处有特异性蛋白条带;氨基酸序列分析证实,我们获得的重组蛋白与人IL-31氨基酸序列一致。(4)注射rhIL-31的小鼠出现频繁的搔抓行为,有脱毛现象,小鼠体重增幅降低;外周血白细胞总数增加,中性粒细胞显著增高,有一定的剂量依赖性。(5)小鼠皮肤病理HE染色可见大量炎性细胞浸润,深达肌层,受损皮肤组织出现棘层肥厚,高剂量实验组可见肌肉溶解。(6)两种注射途径与其对照组比较,小鼠血清细胞因子IL-6、IL-8、L-选择素和MIP-3β水平均有显著增高(p＜0.05),并且随IL-31剂量的增加而增高,具有剂量依赖性;皮下注射实验组IL-6、L-选择素和MIP-3B水平略低于肌肉注射实验组,IL-8水平接近。结论:(1)IPTG浓度为1.0mmol/L,温度37℃左右,诱导5h蛋白表达量最高。(2)用和Ni-NTA琼脂糖凝胶FF柱纯化rhIL-31效果优于sephadex G-75。(3)注射rhIL-31的小鼠体重增幅降低,有频繁的搔抓行为,可见脱毛;外周血白细胞总数增加,中性粒细胞数增加;皮肤病理HE染色见棘层肥厚,大量炎性细胞浸润,高剂量实验组出现肌肉溶解,证明rhIL-31能够诱发小鼠皮肤炎症。(4)小鼠血清细胞因子IL-6、IL-8、L-选择素和MIP-3β水平随rhIL-31剂量的增加而增高,且肌肉注射组高于皮下注射组,表明rhIL-31可能通过诱导表达趋化因子和炎性因子发挥其致炎作用。
[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，

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