本文關(guān)鍵詞：人白細(xì)胞介素22的克隆表達及功能研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年碩士論文　論文類型：學(xué)位論文

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【摘要】：白細(xì)胞介素-22(IL-22)是于2000年發(fā)現(xiàn)的由激活的T細(xì)胞分泌的細(xì)胞因子,屬于IL-10家族。功能性IL-22受體復(fù)合物由IL-22R1和IL-10R2兩條受體鏈組成,IL-22通過與兩條受體鏈的結(jié)合,激活受體復(fù)合物介導(dǎo)的多重信號途徑,包括Janus kinase信號轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄活化蛋白(STAT)的激活劑及在肝細(xì)胞被激活的促有絲分裂蛋白激酶途徑。而與其中一個受體單獨結(jié)合時,IL-22不能介導(dǎo)細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)通路。在功能方面,IL-22可以上調(diào)肝急性期蛋白表達和胰腺相關(guān)蛋白PAP1/Reg2和OPN的高表達,表明IL-22在炎癥反應(yīng)中起作用;在腸上皮細(xì)胞、肺上皮細(xì)胞、神經(jīng)元細(xì)胞、腎小球系膜細(xì)胞、肝細(xì)胞及黑素瘤細(xì)胞中,IL-22能激活STAT轉(zhuǎn)錄因子。它對伴刀豆球蛋白(ConA)刺激下Th2細(xì)胞的IL-4的分泌有一定的抑制作用,這對于哮喘也有潛在的治療作用。近期研究表明,IL-22是肝細(xì)胞的一個存活因子,激活的T細(xì)胞通過釋放IL-22也可以預(yù)防和修復(fù)肝損傷。 本研究從CD3抗體和ConA共刺激的人外周血白細(xì)胞中提取總RNA,利用RT-PCR方法擴增得到了不含信號肽的hIL-22的基因。然后,將hIL-22基因插入pBV220載體中,構(gòu)建重組質(zhì)粒pBV-IL22。同時為了嘗試可溶性表達hIL-22,我們將目的基因插入pET-17b載體中,構(gòu)建了重組質(zhì)粒pET-IL22。將pBV-IL22分別轉(zhuǎn)化大腸桿菌DH5α、JM109、HB101,將pET-IL22轉(zhuǎn)化大腸桿菌BL21,篩選出陽性轉(zhuǎn)化菌落獲得相應(yīng)工程菌株,結(jié)果表明hIL-22在大腸桿菌HB101中表達量最高,可達菌體總蛋白的40%以上。目的蛋白在大腸桿菌中的表達產(chǎn)物以包涵體形式存在,包涵體經(jīng)洗滌后以Sephacryl-300凝膠柱層析純化,目的蛋白的純度可達90%以上。純化蛋白在還原型和氧化型谷胱甘肽組成的氧化還原體系中進行復(fù)性,PEG20000濃縮,獲得了具有活性的hIL-22的純品。MTT法檢測到hIL-22對HepG2細(xì)胞有明顯的促增殖作用。利用半定量RT-PCR方法初步探討了IL-22體外刺激人肝癌細(xì)胞HepG2、正常人肝細(xì)胞LO2表達c-myc和bcl-2的情況,結(jié)
[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 偽, 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
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

【共引文獻】

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1 榮敏;水貂IGF-Ⅰ基因的克隆、表達、功能檢驗及多態(tài)分析[D];中國農(nóng)業(yè)科學(xué)院;2010年

相關(guān)碩士學(xué)位論文 前1條

1 何立麗;VEGFR胞外結(jié)合域（KDR3）基因的克隆表達及功能研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2006年

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本文編號：1410320