Th17细胞相关因子的表达纯化及吸附评价
发布时间：2018-08-24 15:54
【摘要】：自身免疫性疾病是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病,该疾病的发病机理比较复杂,尚未研究清楚,目前临床上还没有有效成熟的治疗方法。之前一直认为自身免疫性疾病与机体内T辅助细胞的两个亚群Th1细胞和Th2细胞有关,当这两个亚群相互作用失去平衡即引起疾病的产生,但是随着研究的深入,发现这种传统观念无法解释一些疾病的发生发展,而相应的一种新的细胞亚群(Th17细胞亚群)的发现能很好的解释许多疑惑,这种新的细胞亚群打破了之前的传统概念,被发现与自身免疫性疾病有着更为密切的关系,近些年来受到了很多研究学者的关注。Th17细胞主要相关效应因子为IL-17、IL-6、TNF-α,从临床上疾病治疗角度出发,关于Th17细胞相关因子的检测与去除具有重要的实际意义。本论文对Th17细胞的主要效应相关因子进行表达纯化,然后通过典型吸附剂对其吸附评价。具体的研究内容有：(1) TNF-α的表达纯化。构建表达载体pET23a-TNF-α,转入宿主BL21 (DE3)中,通过IPTG诱导进行蛋白表达。通过优化条件,得到蛋白最优表达条件为：LB培养基培养菌体,37℃,175 rpm活化12 h,以1%接种量转接放大培养3 h,用0.1mM IPTG诱导培养4 h,目的蛋白的表达量约为1.2 mg/mL,占总蛋白56%。将菌体超声破碎,0.2%PEI (w/w)沉淀,40%饱和硫酸铵沉淀,Ni-NTA亲和树脂层析后,样品纯度达95%以上。(2) IL-6, IL-17A/17F的表达。通过构建表达载体pPIC9K-IL-6, pPIC9K-IL-17A,和pPIC9K-IL-17F,电转入GS115宿主体内,通过RDB培养基,MM/MD培养基,PCR扩增筛选鉴定His+Mut+表型,对阳性转化子用YPD培养基于30℃,225 rpm活化14-16h,以1%接种量转入BMGY培养基于30℃,225 rpm培养至OD600为2-6,接着转入BMMY培养基使OD600为1左右,25℃,225 rpm培养96 h,每24 h甲醇诱导一次,诱导量为终浓度1%,IL-6诱导后表达,IL-17A和17F尚未证实有表达。(3)聚苯乙烯型吸附剂对炎症因子的吸附评价。通过吸附动力学和吸附等温线研究,证实吸附剂对TNF-α吸附速率较快,约2h即接近最大吸附值。聚苯乙烯型吸附剂与TNF-α的亲和常数为57 mL/mg,即9.7×105 L/mol,平衡解离常数(Kd)为0.018mg/mL,最大理论结合容量(Qm)为845.49μg/g,吸附剂对IL-6同样具有吸附性。通过论文研究,获得了Th17细胞主要相关因子的表达纯化方法,探索了典型吸附剂对炎症因子的吸附性能,为自身免疫性疾病的相关研究奠定了良好的基础。
[Abstract]:Autoimmune disease is a disease caused by autoimmune reaction to autoantigen. The pathogenesis of autoimmune disease is complex and has not been studied clearly. At present, there is no effective and mature treatment method in clinic. Autoimmune diseases have long been thought to be associated with two subsets of T helper cells, Th1 cells and Th2 cells, which cause disease when the interaction of these two subsets is out of balance, but as the study progresses, The discovery of a new cell subgroup (Th17 cell subgroup), which cannot explain the occurrence and development of some diseases, is a good explanation for many doubts. This new cell subgroup breaks down the traditional concept. It has been found to be more closely related to autoimmune diseases. In recent years, many researchers have paid close attention to IL-17,IL-6,TNF- 伪, the main effector of Th17 cells, from the point of view of clinical disease treatment. The detection and removal of Th17 cytokines are of great practical significance. In this paper, the main effect-related factors of Th17 cells were expressed and purified, and then evaluated by typical adsorbents. The specific research contents are as follows: (1) expression and purification of TNF- 伪. The expression vector pET23a-TNF- 伪 was constructed and transferred into host BL21 (DE3). The protein was induced by IPTG. The optimum conditions for protein expression were obtained by optimizing the conditions of protein expression, which were activated for 12 h at 37 鈩，
本文编号：2201302
[Abstract]:Autoimmune disease is a disease caused by autoimmune reaction to autoantigen. The pathogenesis of autoimmune disease is complex and has not been studied clearly. At present, there is no effective and mature treatment method in clinic. Autoimmune diseases have long been thought to be associated with two subsets of T helper cells, Th1 cells and Th2 cells, which cause disease when the interaction of these two subsets is out of balance, but as the study progresses, The discovery of a new cell subgroup (Th17 cell subgroup), which cannot explain the occurrence and development of some diseases, is a good explanation for many doubts. This new cell subgroup breaks down the traditional concept. It has been found to be more closely related to autoimmune diseases. In recent years, many researchers have paid close attention to IL-17,IL-6,TNF- 伪, the main effector of Th17 cells, from the point of view of clinical disease treatment. The detection and removal of Th17 cytokines are of great practical significance. In this paper, the main effect-related factors of Th17 cells were expressed and purified, and then evaluated by typical adsorbents. The specific research contents are as follows: (1) expression and purification of TNF- 伪. The expression vector pET23a-TNF- 伪 was constructed and transferred into host BL21 (DE3). The protein was induced by IPTG. The optimum conditions for protein expression were obtained by optimizing the conditions of protein expression, which were activated for 12 h at 37 鈩，
本文编号：2201302
本文链接：https://www.wllwen.com/yixuelunwen/nfm/2201302.html
最近更新
教材专著
