【摘要】：自身免疫性疾病是指機體對自身抗原發(fā)生免疫反應(yīng)而導(dǎo)致自身組織損害所引起的疾病,該疾病的發(fā)病機理比較復(fù)雜,尚未研究清楚,目前臨床上還沒有有效成熟的治療方法。之前一直認(rèn)為自身免疫性疾病與機體內(nèi)T輔助細(xì)胞的兩個亞群Th1細(xì)胞和Th2細(xì)胞有關(guān),當(dāng)這兩個亞群相互作用失去平衡即引起疾病的產(chǎn)生,但是隨著研究的深入,發(fā)現(xiàn)這種傳統(tǒng)觀念無法解釋一些疾病的發(fā)生發(fā)展,而相應(yīng)的一種新的細(xì)胞亞群(Th17細(xì)胞亞群)的發(fā)現(xiàn)能很好的解釋許多疑惑,這種新的細(xì)胞亞群打破了之前的傳統(tǒng)概念,被發(fā)現(xiàn)與自身免疫性疾病有著更為密切的關(guān)系,近些年來受到了很多研究學(xué)者的關(guān)注。Th17細(xì)胞主要相關(guān)效應(yīng)因子為IL-17、IL-6、TNF-α,從臨床上疾病治療角度出發(fā),關(guān)于Th17細(xì)胞相關(guān)因子的檢測與去除具有重要的實際意義。本論文對Th17細(xì)胞的主要效應(yīng)相關(guān)因子進(jìn)行表達(dá)純化,然后通過典型吸附劑對其吸附評價。具體的研究內(nèi)容有：(1) TNF-α的表達(dá)純化。構(gòu)建表達(dá)載體pET23a-TNF-α,轉(zhuǎn)入宿主BL21 (DE3)中,通過IPTG誘導(dǎo)進(jìn)行蛋白表達(dá)。通過優(yōu)化條件,得到蛋白最優(yōu)表達(dá)條件為：LB培養(yǎng)基培養(yǎng)菌體,37℃,175 rpm活化12 h,以1%接種量轉(zhuǎn)接放大培養(yǎng)3 h,用0.1mM IPTG誘導(dǎo)培養(yǎng)4 h,目的蛋白的表達(dá)量約為1.2 mg/mL,占總蛋白56%。將菌體超聲破碎,0.2%PEI (w/w)沉淀,40%飽和硫酸銨沉淀,Ni-NTA親和樹脂層析后,樣品純度達(dá)95%以上。(2) IL-6, IL-17A/17F的表達(dá)。通過構(gòu)建表達(dá)載體pPIC9K-IL-6, pPIC9K-IL-17A,和pPIC9K-IL-17F,電轉(zhuǎn)入GS115宿主體內(nèi),通過RDB培養(yǎng)基,MM/MD培養(yǎng)基,PCR擴增篩選鑒定His+Mut+表型,對陽性轉(zhuǎn)化子用YPD培養(yǎng)基于30℃,225 rpm活化14-16h,以1%接種量轉(zhuǎn)入BMGY培養(yǎng)基于30℃,225 rpm培養(yǎng)至OD600為2-6,接著轉(zhuǎn)入BMMY培養(yǎng)基使OD600為1左右,25℃,225 rpm培養(yǎng)96 h,每24 h甲醇誘導(dǎo)一次,誘導(dǎo)量為終濃度1%,IL-6誘導(dǎo)后表達(dá),IL-17A和17F尚未證實有表達(dá)。(3)聚苯乙烯型吸附劑對炎癥因子的吸附評價。通過吸附動力學(xué)和吸附等溫線研究,證實吸附劑對TNF-α吸附速率較快,約2h即接近最大吸附值。聚苯乙烯型吸附劑與TNF-α的親和常數(shù)為57 mL/mg,即9.7×105 L/mol,平衡解離常數(shù)(Kd)為0.018mg/mL,最大理論結(jié)合容量(Qm)為845.49μg/g,吸附劑對IL-6同樣具有吸附性。通過論文研究,獲得了Th17細(xì)胞主要相關(guān)因子的表達(dá)純化方法,探索了典型吸附劑對炎癥因子的吸附性能,為自身免疫性疾病的相關(guān)研究奠定了良好的基礎(chǔ)。
[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- 偽, 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- 偽. The expression vector pET23a-TNF- 偽 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，

本文編號：2201302