人白细胞介素22的克隆表达及功能研究

发布时间：2018-01-11 16:28

本文关键词：人白细胞介素22的克隆表达及功能研究　出处：《中国人民解放军军事医学科学院》2005年硕士论文　论文类型：学位论文

【摘要】：白细胞介素-22(IL-22)是于2000年发现的由激活的T细胞分泌的细胞因子,属于IL-10家族。功能性IL-22受体复合物由IL-22R1和IL-10R2两条受体链组成,IL-22通过与两条受体链的结合,激活受体复合物介导的多重信号途径,包括Janus kinase信号转导及转录活化蛋白(STAT)的激活剂及在肝细胞被激活的促有丝分裂蛋白激酶途径。而与其中一个受体单独结合时,IL-22不能介导细胞内信号转导通路。在功能方面,IL-22可以上调肝急性期蛋白表达和胰腺相关蛋白PAP1/Reg2和OPN的高表达,表明IL-22在炎症反应中起作用;在肠上皮细胞、肺上皮细胞、神经元细胞、肾小球系膜细胞、肝细胞及黑素瘤细胞中,IL-22能激活STAT转录因子。它对伴刀豆球蛋白(ConA)刺激下Th2细胞的IL-4的分泌有一定的抑制作用,这对于哮喘也有潜在的治疗作用。近期研究表明,IL-22是肝细胞的一个存活因子,激活的T细胞通过释放IL-22也可以预防和修复肝损伤。本研究从CD3抗体和ConA共刺激的人外周血白细胞中提取总RNA,利用RT-PCR方法扩增得到了不含信号肽的hIL-22的基因。然后,将hIL-22基因插入pBV220载体中,构建重组质粒pBV-IL22。同时为了尝试可溶性表达hIL-22,我们将目的基因插入pET-17b载体中,构建了重组质粒pET-IL22。将pBV-IL22分别转化大肠杆菌DH5α、JM109、HB101,将pET-IL22转化大肠杆菌BL21,筛选出阳性转化菌落获得相应工程菌株,结果表明hIL-22在大肠杆菌HB101中表达量最高,可达菌体总蛋白的40%以上。目的蛋白在大肠杆菌中的表达产物以包涵体形式存在,包涵体经洗涤后以Sephacryl-300凝胶柱层析纯化,目的蛋白的纯度可达90%以上。纯化蛋白在还原型和氧化型谷胱甘肽组成的氧化还原体系中进行复性,PEG20000浓缩,获得了具有活性的hIL-22的纯品。MTT法检测到hIL-22对HepG2细胞有明显的促增殖作用。利用半定量RT-PCR方法初步探讨了IL-22体外刺激人肝癌细胞HepG2、正常人肝细胞LO2表达c-myc和bcl-2的情况,结
[Abstract]:Interleukin-22 (IL-22) is a cytokine secreted by activated T cells in 2000. Functional IL-22 receptor complex is composed of two receptor chains, IL-22R1 and IL-10R2, by binding to two receptor chains. Activation of multiple signaling pathways mediated by receptor complexes. These include activators of Janus kinase signal transduction and activator of transcription protein (STAT) and mitogen-stimulating protein kinase pathway activated in hepatocytes. IL-22 can not mediate intracellular signal transduction pathway. IL-22 can up-regulate the expression of protein in acute phase of liver and the high expression of PAP1/Reg2 and OPN in pancreas. It is suggested that IL-22 plays an important role in inflammatory reaction. In intestinal epithelial cells, lung epithelial cells, neuronal cells, glomerular Mesangial cells, hepatocytes, and melanoma cells. IL-22 can activate STAT transcription factor. It can inhibit the secretion of IL-4 in Th2 cells stimulated by concanavalin Cona. Recent studies have shown that IL-22 is a survival factor in hepatocytes and activated T cells can also prevent and repair liver injury by releasing IL-22. In this study, total RNA was extracted from human peripheral blood leukocytes co-stimulated with CD3 antibody and ConA. The hIL-22 gene without signal peptide was amplified by RT-PCR. Then, the hIL-22 gene was inserted into the pBV220 vector. The recombinant plasmid pBV-IL22 was constructed and the target gene was inserted into the pET-17b vector in order to try to express hIL-22 in a soluble way. The recombinant plasmid pET-IL22 was constructed. The recombinant plasmid pET-IL22 was transformed into E. coli DH5 伪, JM109 and HB101, respectively, and the pET-IL22 was transformed into Escherichia coli BL21. The positive transformed colonies were screened and the corresponding engineering strains were obtained. The results showed that hIL-22 expression was the highest in Escherichia coli HB101. The expression product of the target protein in Escherichia coli existed as inclusion body, and the inclusion body was purified by Sephacryl-300 gel column chromatography after washing. The purity of the purified protein was over 90%. The purified protein was concentrated in the redox system composed of reduced and oxidized glutathione. The purified hIL-22 with activity was obtained. MTT assay showed that hIL-22 could significantly promote the proliferation of HepG2 cells. A preliminary study of IL- was carried out by semi-quantitative RT-PCR method. Human hepatoma cell line HepG2 was stimulated in vitro. Expression of c-myc and bcl-2 in LO2 of normal human hepatocytes
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

【共引文献】