本文關(guān)鍵詞： IL-17 Th17 融合蛋白 包涵體 復(fù)性 HeLa　出處：《蘇州大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： Th1-Th2的經(jīng)典分類的提出已經(jīng)有20年了，它為我們深入理解固有免疫和適應(yīng)性免疫之間的相互聯(lián)系提供了新的思路和研究模型。然而隨著IL-17和IL-12兩個(gè)家族的細(xì)胞因子的發(fā)現(xiàn)和研究，這種分類方法逐漸被予以改變：認(rèn)為機(jī)體內(nèi)存在以分泌IL-17為特征的T細(xì)胞亞群，和以往的Th1，Th2都不同，這群細(xì)胞在IL-23，IL-6和IL-1誘導(dǎo)后產(chǎn)生IL-17細(xì)胞因子，并發(fā)揮獨(dú)特的生物學(xué)效應(yīng)，從而解釋了以前在免疫調(diào)節(jié)，宿主抵抗病菌，免疫病理等方面未能解釋的問(wèn)題。這種T細(xì)胞現(xiàn)在命名為Th17細(xì)胞。它的特異性在于能分泌IL-17細(xì)胞因子，參與體內(nèi)多種免疫反應(yīng)。Th17細(xì)胞數(shù)量和IL-17在機(jī)體內(nèi)表達(dá)水平的改變，以及Th17細(xì)胞和Th1細(xì)胞比例的變化與疾病的發(fā)展密切相關(guān)并成為廣為關(guān)注的研究課題。為此，本研究旨在表達(dá)重組蛋白和生物學(xué)特性分析作為切入點(diǎn)，為本單位開(kāi)展相關(guān)工作奠定了一定的物質(zhì)基礎(chǔ)。 一．人IL-17基因全長(zhǎng)及去信號(hào)肽片段的克隆和載體的構(gòu)建 根據(jù)文獻(xiàn)報(bào)道的人IL-47的基因序列設(shè)計(jì)合成了全長(zhǎng)和去信號(hào)肽的特異性引物。采用RT-PCR方法從人外周血PHA刺激活化的T細(xì)胞中擴(kuò)增了人IL-17全長(zhǎng)基因和去信號(hào)肽基因。應(yīng)用雙酶切法酶切載體和目的基因，再用連接酶連接酶切回收后的產(chǎn)物。測(cè)序正確后，在TOP10宿主菌中增殖PQE3.0／IL-17和PCEP4／IL-17質(zhì)粒，經(jīng)酶切和PCR鑒定后PQE3.0／IL-17轉(zhuǎn)化M15表達(dá)菌株。 二．人PQE3.0／IL-17融合蛋白在大腸桿菌中的表達(dá)和鑒定 在LB細(xì)菌培養(yǎng)基中增殖M15表達(dá)菌，采取一系列實(shí)驗(yàn)條件，包括降低溫度，縮短誘導(dǎo)時(shí)間等，均證實(shí)不能誘導(dǎo)可溶性IL-17融合蛋白的表達(dá)，而表達(dá)的蛋白主要集中在包涵體內(nèi)。表達(dá)動(dòng)力學(xué)分析表明，應(yīng)用1 mmol／L IPTG誘導(dǎo)5 h可獲得最有效的表達(dá)。實(shí)驗(yàn)室規(guī)模增殖表達(dá)菌，經(jīng)超聲裂解和離心，沉淀變性及復(fù)性，透析后經(jīng)HiTrap親和層析柱一步純化融合蛋白。實(shí)驗(yàn)結(jié)果表明，純化后的IL-17／His融合蛋白純度可達(dá)90％以上，定量為2 mg／ml。應(yīng)用Western-blot對(duì)融合蛋白進(jìn)行生物學(xué)鑒定，結(jié)果為約15KD的單一條帶。 三．人IL-17在真核系統(tǒng)中的表達(dá) 抽取PCEP4／IL-17質(zhì)粒，轉(zhuǎn)染CHO細(xì)胞，應(yīng)用潮霉素進(jìn)行篩選，在顯微鏡下觀察到陽(yáng)性克隆，胰蛋白酶消化細(xì)胞，再用移液尖挑取陽(yáng)性克隆分孔培養(yǎng)，繼續(xù)使用藥物篩選，直到所獲得的克隆表達(dá)穩(wěn)定為止。吸取陽(yáng)性克隆細(xì)胞培養(yǎng)的上清，用ELISA方法定性和定量檢測(cè)IL-17的表達(dá)情況。實(shí)驗(yàn)的結(jié)果是所獲得表達(dá)量較高的融合蛋白。 四．人IL-17重組蛋白的生物學(xué)功能的初步研究 采用人的宮頸癌細(xì)胞株HeLa作為反應(yīng)細(xì)胞，按不同濃度加入IL-17／His融合蛋白和IL-17／Fc融合蛋白，反應(yīng)48小時(shí)后，用ELISA法檢測(cè)上清IL-6和GM-CSF的水平，與對(duì)照組作比較，觀察兩種IL-17蛋白對(duì)HeLa細(xì)胞的作用在一定劑量范圍內(nèi)是否存在劑量依賴關(guān)系以及兩種蛋白活性度的高低。 綜上所述，本項(xiàng)研究我們成功地應(yīng)用原核和真核系統(tǒng)表達(dá)出了人IL-17重組蛋白，并進(jìn)行了生物學(xué)活性的初步研究。所表達(dá)的蛋白具有一定的生物學(xué)活性，為我們下一步工作奠定了基礎(chǔ)。涉及IL-17錨定蛋白和單克隆抗體的研制工作尚待下一步繼續(xù)展開(kāi)。
[Abstract]:The classic Th1-Th2 classification has been proposed for 20 years, which provides new ideas and research model for us to further understand the relationship between innate and adaptive immunity. However with cytokines IL-17 and IL-12 in two families, the discovery and research of this classification method has been changed to that body to the secretion of IL-17 of T cell subsets, and the previous Th1, Th2 are different, this group of cells in IL-23, IL-17, IL-6 and IL-1 induced cytokine production, and play biological effects unique, thus explaining the former in immune regulation, immune host defense against pathogen, pathology and other aspects of the problem. This can not explain T cells now named Th17 cells. Its specificity is IL-17 can secrete cytokines, participate in the change of expression level and the number of IL-17.Th17 cells in vivo immune response in the body, and Th17 The change of cell to Th1 cell ratio is closely related to the development of disease and has become a research topic of widespread concern. Therefore, the purpose of this study is to express recombinant protein and biological characteristics as a breakthrough point, laying a solid foundation for the related work of our unit.
The cloning and construction of the full length of human IL-17 gene and the fragment of the de signal peptide
According to the length and specific primers were designed and synthesized to signal peptide gene sequences reported in the literature. IL-47 from human peripheral blood PHA stimulated by RT-PCR method in T cells amplified human IL-17 full-length gene and signal peptide gene. The application of double enzyme digestion enzyme carrier and target genes, and ligase the digestion products recovered. Ligase after sequencing, the TOP10 host cell proliferation of PQE3.0 / IL-17 and PCEP4 / IL-17 plasmids were identified by enzyme digestion and PCR after PQE3.0 / IL-17 conversion M15 expression strain.
Two. Expression and identification of human PQE3.0 / IL-17 fusion protein in Escherichia coli
The expression of M15 in bacteria proliferation medium LB bacterial culture, take a series of experimental conditions, including lowering the temperature, shorten the induction time, confirmed the expression of soluble IL-17 fusion protein could not be induced, and the expression of the protein is mainly concentrated in the inclusion body. The expression kinetics analysis shows that the application of 1 mmol / L 5 h induced IPTG expression can be obtained the most effective laboratory scale. The proliferation of expression of bacteria, after ultrasonic lysis and centrifugation, precipitation denaturation and renaturation after dialysis by HiTrap affinity chromatography purified fusion protein. The experimental results show that the purified IL-17 / His fusion protein reached a purity of more than 90%, 2 mg / ml. for the quantitative application of Western-blot fusion protein the biological identification results for a single band of about 15KD.
Three. Expression of human IL-17 in the eukaryotic system
Extraction of PCEP4 / IL-17 plasmid was transfected into CHO cells using hygromycin screening, positive clones were observed under the microscope, trypsin digestion and pipette tip cells, positive clones with hole culture, continue to use drug screening, cloning and expression of the stable until now. Pipeted the cell culture supernatant, with the expression of ELISA method for qualitative and quantitative detection of IL-17. The experimental results are obtained by the high expression of fusion protein.
Four. Preliminary study on the biological function of human IL-17 recombinant protein
The human cervical cancer cell line HeLa as the reaction cells with different concentration adding IL-17 / His fusion protein and IL-17 / Fc fusion protein, 48 hours after the reaction, ELISA was used to detect the supernatant of IL-6 and GM-CSF levels, compared with the control group, to observe the effect of two kinds of IL-17 protein in HeLa cells in a certain dose in the range of dose dependent and two kinds of high and low protein activity.
In summary, in this study we successfully used prokaryotic and eukaryotic expression system. Recombinant human IL-17 protein, and studied the biological activity. The expression protein has certain biological activity, for our next work laid the foundation for development work. Relates to IL-17 anchored protein and monoclonal antibody is to be the next step to continue to expand.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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本文編號(hào)：1481279