本文關(guān)鍵詞：人分化抑制因子Id-2、Id-3在E.coli中的高效表達(dá)及其多克隆抗體的制備　出處：《黑龍江大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人分化抑制因子2(Id-2) 人分化抑制因子3(Id-3) 表達(dá) 純化 多克隆抗體

【摘要】：分化抑制因子又稱DNA結(jié)合抑制因子,屬于螺旋-環(huán)-螺旋蛋白,其對堿性螺旋-環(huán)-螺旋轉(zhuǎn)錄因子有負(fù)調(diào)控作用,可以抑制細(xì)胞分化、促進(jìn)細(xì)胞增殖并且參與細(xì)胞周期調(diào)控過程。目前已知哺乳動物細(xì)胞含有4種亞型Id蛋白即Id-1~4。其中Id-2在控制細(xì)胞分化和增殖過程中發(fā)揮著重要的作用。它不但能對肝細(xì)胞轉(zhuǎn)錄因子進(jìn)行負(fù)調(diào)節(jié),抑制肝星狀細(xì)胞分化,而且有研究表明Id-2在凋亡誘導(dǎo)的肝壞死中也扮演著重要角色。另外,Id-2表達(dá)隨細(xì)胞衰老呈明顯下降趨勢,在破骨細(xì)胞、乳腺上皮、成纖維、角質(zhì)、內(nèi)皮等細(xì)胞中發(fā)現(xiàn),其過度表達(dá)可以延遲細(xì)胞老化或?qū)е录?xì)胞永生化。Id-2基因也具有癌基因的特性,其編碼的Id-2蛋白能阻止細(xì)胞分化,促進(jìn)細(xì)胞增殖,從而參與腫瘤的發(fā)生。而Id-3基因作為血清誘導(dǎo)的立即早期基因,在小鼠成纖維細(xì)胞系中被首次發(fā)現(xiàn),其編碼的Id-3蛋白能夠參與多種細(xì)胞生物學(xué)過程,包括T細(xì)胞和B細(xì)胞的發(fā)育、骨骼肌的分化、血管平滑肌的增值、胚胎的神經(jīng)形成、骨發(fā)生和腫瘤誘導(dǎo)的血管發(fā)生等。目前,Id蛋白已成為研究細(xì)胞生命過程及探尋治療人類疾病有效靶向藥物的一類重要分子。 本研究在大腸桿菌中分別表達(dá)人Id-2與谷胱甘肽-S轉(zhuǎn)移酶(GST)的融合蛋白及人Id-3與谷胱甘肽-S轉(zhuǎn)移酶(GST)的融合蛋白,并在此基礎(chǔ)上制備了抗人Id-2、Id-3的多克隆抗體。本試驗從人乳腺癌組織中提取總RNA,應(yīng)用RT-PCR方法擴(kuò)增出Id-2、Id-3基因的編碼序列,鑒定正確后將其克隆至表達(dá)載體pGEX-6P-1中,重組質(zhì)粒經(jīng)PCR、酶切、測序鑒定后,在大腸桿菌中經(jīng)IPTG誘導(dǎo)表達(dá)獲得GST-Id-2、GST-Id-3融合蛋白,SDS-PAGE分析表達(dá)產(chǎn)物。Western-Blot實驗表明,通過親和層析法純化的GST-Id-2、GST-Id-3融合蛋白具有良好的免疫原性,然后以純化的蛋白為抗原免疫健康新西蘭白兔制備多克隆抗體,并利用酶聯(lián)免疫吸附試驗(ELISA)及瓊脂免疫擴(kuò)散試驗檢測抗體效價。 本試驗結(jié)果表明,經(jīng)PCR、酶切、測序鑒定證明,Id-2、Id-3基因分別已正確克隆至pGEX-6P-1中,經(jīng)IPTG誘導(dǎo)后,表達(dá)出相對分子質(zhì)量為40 000的GST-Id-2融合蛋白及相對分子質(zhì)量為39 000的GST-Id-3融合蛋白。Western-Blot、ELISA和瓊脂雙向擴(kuò)散實驗鑒定所制備的多克隆抗體可以與GST-Id-2和GST-Id-3發(fā)生特異性反應(yīng)。 本試驗研究結(jié)果證明,Id-2、Id-3基因在大腸桿菌中獲得了成功表達(dá)且成功制備了其相應(yīng)的多克隆抗體,為檢測Id-2、Id-3及其在各種組織中的表達(dá)提供了一種檢測方法,也為分析Id-2、Id-3分子結(jié)構(gòu)及抗原表位奠定了基礎(chǔ)。
[Abstract]:The differentiation inhibitory factor called DNA binding inhibitory factor, which belongs to helix loop helix proteins, the basic helix loop helix transcription factor has a negative regulatory role, can inhibit cell differentiation and promote cell proliferation and cell cycle regulation. Known mammalian cells contain 4 subtypes of Id protein in Id-1~4. Id-2 the control of cell differentiation and proliferation plays an important role. It can not only negatively regulate transcription factor on liver cells, inhibiting hepatic stellate cell differentiation, and studies have shown that Id-2 induced apoptosis in hepatic necrosis also play an important role. In addition, the expression of Id-2 with cell senescence was significantly decreased in the osteoclasts, mammary epithelial cells, fibroblasts, keratinocytes, endothelial cells found, its overexpression can delay the aging of cells or cause characteristics of immortalized cell.Id-2 gene with cancer gene, its coding Code Id-2 protein can prevent cell differentiation, promote cell proliferation, and thereby involves in carcinogenesis. The Id-3 gene as immediate early genes induced by serum, a fibroblast cell line was first discovered in mice, its encoding Id-3 protein involved in many cellular processes, including T cell and B cell development, differentiation of bone muscle, vascular smooth muscle proliferation, embryonic neurogenesis, osteogenesis and tumor induced angiogenesis. At present, Id protein has become the research of cellular processes and explore the treatment of human diseases effectively targeted a kind of important molecular drugs.
This study were expressed in Escherichia coli Id-2 and glutathione -S transferase (GST) fusion protein and Id-3 and glutathione -S transferase (GST) fusion protein, and on the basis of the preparation of anti human Id-2 polyclonal antibody of Id-3. The total RNA was extracted from human breast cancer tissues the application of RT-PCR, amplified Id-2, encoding Id-3 gene sequence, after the identification of cloned into expression vector pGEX-6P-1. The recombinant plasmid was identified by PCR, enzyme digestion and sequencing, in Escherichia coli induced by IPTG expression of GST-Id-2, GST-Id-3 fusion protein, SDS-PAGE analysis showed that the expression product of.Western-Blot by affinity experiment. Chromatography purified GST-Id-2 GST-Id-3 fusion protein has good immunogenicity, and then purified protein antigen to immunize healthy New Zealand white rabbits to prepare polyclonal antibody, using enzyme-linked immunosorbent assay (ELISA) and Agar immunodiffusion test was used to detect antibody titer.
The test results show that by PCR, enzyme digestion and sequencing proved that Id-2 Id-3 gene was correctly cloned into pGEX-6P-1. After induced by IPTG, expression of the relative molecular mass of 40000 GST-Id-2 fusion protein and the relative molecular mass of 39000 GST-Id-3 fusion protein.Western-Blot, ELISA polyclonal antibody and double agar diffusion experiment identification of the prepared can react specifically with GST-Id-2 and GST-Id-3.
The test results show that the Id-2 Id-3 gene, obtained the corresponding polyclonal antibody was prepared successfully and successfully expressed in Escherichia coli, for the detection of Id-2, Id-3 and its expression in various tissues and provides a detection method for Id-2 analysis, Id-3 molecular structure and epitope laid the foundation.

【學(xué)位授予單位】：黑龍江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 陳興書,姚忠祥,劉建軍,陳建芳,楊輝,蔡文琴;Id2在成年大鼠喙側(cè)神經(jīng)干細(xì)胞遷移流通道的表達(dá)[J];第三軍醫(yī)大學(xué)學(xué)報;2005年10期

2 高宇;陳興書;余洪俊;劉宏亮;姚忠祥;;DNA結(jié)合抑制因子2在成年大鼠脊髓表達(dá)的研究[J];第三軍醫(yī)大學(xué)學(xué)報;2007年07期

3 李曉軍,秦浚川;細(xì)胞分化抑制因子(Id)研究進(jìn)展[J];生物化學(xué)與生物物理進(jìn)展;2004年10期

4 陳長征,段治軍,楊新穎,夏其昌,李伯良,王應(yīng)睞;T7啟動子控制下的多功能大腸桿菌表達(dá)系統(tǒng)[J];生物化學(xué)與生物物理學(xué)報;1996年05期

5 陳艷艷;張聲;江忠清;;分化抑制因子及其與腫瘤性血管生成[J];國際病理科學(xué)與臨床雜志;2005年06期

6 于新鳳,劉芝華;Id蛋白與腫瘤關(guān)系的研究進(jìn)展[J];世界華人消化雜志;2005年07期

7 李文煉;盧輝山;王川;;ID蛋白與腫瘤關(guān)系的研究進(jìn)展[J];醫(yī)學(xué)綜述;2007年07期

8 Robert Wai-sui LIM;;Molecular mechanisms regulating expression and function of transcription regulator “inhibitor of differentiation 3”[J];Acta Pharmacologica Sinica;2005年12期

，

本文編號：1401078