【摘要】： Foxp3是FOX(forkhead box)家族轉(zhuǎn)錄因子中的一員,是調(diào)節(jié)性T細(xì)胞(regulatory T cells,Treg)的特異性標(biāo)志,同時(shí)對(duì)Treg的發(fā)生、發(fā)育、功能的維持與發(fā)揮等方面起著關(guān)鍵作用。研究發(fā)現(xiàn)表達(dá)外源性Foxp3可使非調(diào)節(jié)性T細(xì)胞具有類似調(diào)節(jié)性T細(xì)胞樣的表型及免疫抑制功能。Foxp3的突變可能導(dǎo)致調(diào)節(jié)性的CD4~+CD25~+T細(xì)胞介導(dǎo)的免疫耐受缺陷。 HIV-1(human immunodeficiency virus type 1)基因編碼的反式激活蛋白(transactivator,TAT)中的蛋白轉(zhuǎn)導(dǎo)結(jié)構(gòu)域(protein transductiondomain,PTD)能夠攜帶外源性蛋白穿入幾乎所有的真核細(xì)胞膜而進(jìn)入細(xì)胞漿和細(xì)胞核內(nèi),同時(shí)具有高效、快速的特點(diǎn)。 目的:構(gòu)建帶HIV-1穿膜序列原核表達(dá)載體pET28a-PTD-△Foxp3、pET28a-PTD-eGFP-△Foxp3、pET28a-PTD-eGFP-Foxp3,表達(dá)并純化小鼠PTD-△Foxp3、PTD-eGFP-△Foxp3、PTD-eGFP-Foxp3融合蛋白,研究融合蛋白導(dǎo)入細(xì)胞的效率及其對(duì)T細(xì)胞活化增殖的免疫調(diào)節(jié)功能的影響。 方法:利用基因重組技術(shù)構(gòu)建pET28a-PTD-△Foxp3、pET28a-PTD-eGFP-△Foxp3、pET28a-PTD-eGFP-Foxp3原核表達(dá)載體,并在大腸埃希菌Rosetta(DE3)中表達(dá)融合蛋白,經(jīng)Ni~(2+)分離柱純化融合蛋白。Western-blot技術(shù)檢測(cè)融合蛋白表達(dá)的正確性,流式細(xì)胞術(shù)檢測(cè)融合蛋白穿越細(xì)胞膜進(jìn)入小鼠T淋巴細(xì)胞瘤株EL-4細(xì)胞中的能力,并通過(guò)混合淋巴細(xì)胞反應(yīng)初步分析融合蛋白對(duì)T細(xì)胞活化增殖的影響。 結(jié)果:成功構(gòu)建了pET28a-PTD-△Foxp3、pET28a-PTD-eGFP-△Foxp3、pET28a-PTD-eGFP-Foxp3融合蛋白表達(dá)載體,表達(dá)并純化了PTD-△Foxp3、PTD-eGFP-△Foxp3、PTD-eGFP-Foxp3融合蛋白。通過(guò)流式細(xì)胞術(shù)分析證實(shí)表達(dá)的融合蛋白均能高效的轉(zhuǎn)入細(xì)胞內(nèi);同時(shí)經(jīng)混合淋巴細(xì)胞反應(yīng)初步表明PTD-eGFP-Foxp3融合蛋白能明顯抑制T淋巴細(xì)胞的活化增殖能力,而PTD-△Foxp3、PTD-eGFP-△Foxp3突變體融合蛋白抑制T細(xì)胞活化增殖的能力喪失。 結(jié)論:成功表達(dá)具有生物學(xué)活性的PTD-△Foxp3、PTD-eGFP-△Foxp3、PTD-eGFP-Foxp3融合蛋白,為進(jìn)一步更好地研究Foxp3的免疫抑制功能和構(gòu)建表達(dá)人PTD-Foxp3相關(guān)融合蛋白奠定了基礎(chǔ)。
[Abstract]:Foxp3 is a member of FOX (forkhead box) family transcription factors and a specific marker of regulatory T cell (regulatory T cells,Treg. It also plays a key role in the genesis, development, function maintenance and development of Treg. It has been found that the expression of exogenous Foxp3 can make non-regulatory T cells have a regulatory T-cell-like phenotype and immunosuppressive function. The mutation of Foxp3 may lead to regulatory CD4~ CD25~ T cell-mediated immune tolerance defects. HIV-1 (human immunodeficiency virus type 1) the protein transduction domain (protein transductiondomain,PTD) of the transactivator protein (transactivator,TAT) encoded by the gene can carry foreign proteins into almost all eukaryotic membranes and into the cytoplasm and nucleus. At the same time, it has the characteristics of high efficiency and fast. Objective: to construct a prokaryotic expression vector pET28a-PTD- Foxp3,pET28a-PTD-eGFP- Foxp3,pET28a-PTD-eGFP-Foxp3, with HIV-1 transmembrane sequence and purify mouse PTD- Foxp3,PTD-eGFP- Foxp3,PTD-eGFP-Foxp3 fusion protein. To study the effect of fusion protein on T cell activation and proliferation. Methods: the prokaryotic expression vector of pET28a-PTD- Foxp3,pET28a-PTD-eGFP- Foxp3,pET28a-PTD-eGFP-Foxp3 was constructed by gene recombination technique, and the fusion protein was expressed in Escherichia coli Rosetta (DE3). The fusion protein was purified by Ni~ (2) column. The expression of fusion protein was detected by Western-blot, and the ability of fusion protein to cross cell membrane into mouse T lymphocyte tumor EL-4 cell line was detected by flow cytometry. The effect of fusion protein on T cell activation and proliferation was analyzed by mixed lymphocyte reaction. Results: pET28a-PTD- Foxp3,pET28a-PTD-eGFP- Foxp3,pET28a-PTD-eGFP-Foxp3 fusion protein expression vector was successfully constructed, and PTD- Foxp3,PTD-eGFP- Foxp3,PTD-eGFP-Foxp3 fusion protein was expressed and purified. Flow cytometry showed that the expressed fusion protein could be efficiently transferred into the cells. At the same time, the mixed lymphocyte reaction showed that PTD-eGFP-Foxp3 fusion protein could significantly inhibit the activation and proliferation of T lymphocytes, while the PTD- Foxp3,PTD-eGFP- Foxp3 fusion protein could inhibit the activation and proliferation of T cells. Conclusion: the successful expression of PTD- Foxp3,PTD-eGFP- Foxp3,PTD-eGFP-Foxp3 fusion protein with biological activity lays a foundation for the further study of the immunosuppressive function of Foxp3 and the construction of human PTD-Foxp3 related fusion protein.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R341

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳忠東,馬艷,曾憲端;妊娠滋養(yǎng)葉細(xì)胞疾病p21蛋白表達(dá)的研究[J];癌癥;1999年S1期

2 李治昊;高梅;葉珊;趙京卉;甘友清;;蛋白質(zhì)轉(zhuǎn)導(dǎo)技術(shù)的應(yīng)用及研究進(jìn)展[J];西部醫(yī)學(xué);2007年06期

3 胡晨;王越;陳彬;姜曉梅;李渝萍;婁桂予;張艷;何鳳田;周度金;;PNRC多肽通過(guò)干擾核受體途徑抑制BT474細(xì)胞的增殖[J];現(xiàn)代腫瘤醫(yī)學(xué);2010年06期

4 陳彬;王越;胡晨;陽(yáng)玉鵬;李渝萍;婁桂予;張艷;何鳳田;周度金;;PNRC多肽通過(guò)影響Ras信號(hào)途徑抑制BT-474細(xì)胞的增殖[J];中國(guó)癌癥雜志;2009年03期

5 ;[J];;年期

6 ;[J];;年期

7 ;[J];;年期

8 ;[J];;年期

9 ;[J];;年期

10 ;[J];;年期

相關(guān)會(huì)議論文 前3條

1 張晨;陳曉超;劉樹(shù)滔;劉元?jiǎng)?饒平凡;;融合蛋白PTD-SOD對(duì)紫外輻射下L-02細(xì)胞的保護(hù)和修復(fù)能力的研究[A];中國(guó)食品科學(xué)技術(shù)學(xué)會(huì)第五屆年會(huì)暨第四屆東西方食品業(yè)高層論壇論文摘要集[C];2007年

2 劉樹(shù)滔;張晨;趙花琴;陳曉超;饒平凡;;融合蛋白PTD-SOD對(duì)紫外輻射引起的細(xì)胞損傷的保護(hù)作用[A];中國(guó)食品科學(xué)技術(shù)學(xué)會(huì)第五屆年會(huì)暨第四屆東西方食品業(yè)高層論壇論文摘要集[C];2007年

3 胡晨;王越;陳彬;姜曉梅;李渝萍;婁桂予;張艷;何鳳田;周度金;;PNRC多肽通過(guò)干擾核受體途徑抑制BT474細(xì)胞的增殖[A];重慶市生物化學(xué)與分子生物學(xué)學(xué)術(shù)會(huì)議論文摘要匯編[C];2009年

相關(guān)重要報(bào)紙文章 前3條

1 吳曼;中國(guó)檢測(cè)技術(shù)堪稱一流[N];中國(guó)國(guó)門(mén)時(shí)報(bào);2002年

2 記者 董惠池;中美UL安全認(rèn)證合作成果顯著[N];中國(guó)國(guó)門(mén)時(shí)報(bào)(中國(guó)出入境檢驗(yàn)疫報(bào));2000年

3 特約記者 左世云;中國(guó)石油技術(shù)開(kāi)發(fā)公司出口土庫(kù)曼第一套鉆機(jī)成功投產(chǎn)[N];中國(guó)石油報(bào);2002年

相關(guān)博士學(xué)位論文 前1條

1 張才;NPY對(duì)奶牛肝糖異生和脂肪動(dòng)員關(guān)鍵酶表達(dá)的影響[D];吉林大學(xué);2008年

相關(guān)碩士學(xué)位論文 前2條

1 張美芳;PTD-GFP、PTD-EDAG融合蛋白質(zhì)的表達(dá)、純化和初步應(yīng)用[D];天津大學(xué);2008年

2 周小兵;PTD-ΔhNFATminiDBD蛋白在大鼠心臟移植急性排斥模型中的應(yīng)用研究[D];江蘇大學(xué);2010年

，

本文編號(hào)：2407739