本文選題：原核表達(dá) + 多克隆抗血清��； 參考：《第四軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 細(xì)胞遷移指的是細(xì)胞在接收到遷移信號或感受到某些物質(zhì)的濃度梯度后而產(chǎn)生的移動。趨化因子作為一種信號分子通過與細(xì)胞膜表面上的受體結(jié)合,啟動細(xì)胞內(nèi)信號,激活或抑制肌動蛋白結(jié)合蛋白的活性,最終改變細(xì)胞骨架的狀態(tài),引起細(xì)胞的移動。基質(zhì)細(xì)胞衍生因子-1(Stromal cell derived factor-1,SDF-1)就是一種重要的趨化因子,廣泛地表達(dá)于各種細(xì)胞和組織中,包括免疫細(xì)胞、腦、心臟、腎、肝、肺和脾,在免疫系統(tǒng)、循環(huán)系統(tǒng)及中樞神經(jīng)系統(tǒng)的發(fā)育中起著至關(guān)重要的作用。同時與HIV感染、炎癥反應(yīng)、造血細(xì)胞的調(diào)控作用、腫瘤細(xì)胞的遷移等密切相關(guān)。歷來認(rèn)為,SDF-1是通過CXCR4這個唯一的受體來調(diào)控這些不同的過程。但近來發(fā)現(xiàn)了SDF-1的一個新受體,稱為CXCR7。CXCR7表達(dá)于許多腫瘤細(xì)胞系,活化的內(nèi)皮細(xì)胞以及胎肝細(xì)胞,大多數(shù)正常細(xì)胞表面不表達(dá),盡管這些正常細(xì)胞表面不表達(dá)CXCR7分子,但其細(xì)胞內(nèi)卻表達(dá)了CXCR7 mRNA。目前的研究表明在體外CXCR7具有促進(jìn)細(xì)胞存活的作用,并對小鼠腫瘤生長有促進(jìn)作用,但關(guān)于SDF-1/CXCR7對細(xì)胞遷移的作用并不明確,我們進(jìn)行了如下兩個方面的研究: 1、SDF-1α的原核表達(dá)。用已有的pMD-18T-SDF-1α質(zhì)粒與pGEX-4T-1表達(dá)載體構(gòu)建融合表達(dá)載體pGEX-4T-1-SDF-1α,并在大腸桿菌BL21中進(jìn)行誘導(dǎo)表達(dá)。確定目的基因的表達(dá)情況后,進(jìn)一步大量表達(dá),利用Glutathione Sepharose 4B柱,對獲得的融合蛋白進(jìn)行純化。2、CXCR7抗血清的制備、鑒定以及對MCF-7細(xì)胞遷移影響。合成兩條肽段H458、H459,和KLH交聯(lián)后免疫新西蘭兔,制備CXCR7的多克隆抗血清,飽和硫酸胺法純化,酶聯(lián)免疫反應(yīng)(ELISA)檢測抗血清滴度, Western-blot和間接免疫熒光檢測多克隆抗體對CXCR7受體的識別能力。采用Millicell小室來研究SDF-1/CXCR7對細(xì)胞遷移的影響,分別對抗體封閉組、趨化因子組和對照組的遷移細(xì)胞進(jìn)行計數(shù)。 結(jié)果:雙酶切鑒定證明我們成功地構(gòu)建了重組融合表達(dá)載體pGEX-4T-1-SDF-1α,IPTG誘導(dǎo)融合蛋白GST-SDF-1α表達(dá),主要以包涵體的形式存在。對包涵體充分洗滌后,利用Glutathione Sepharose 4B柱,獲得了純化的GST-SDF-1α融合蛋白。用與KLH交聯(lián)的肽段H458、H459免疫新西蘭兔,獲得多克隆抗血清,間接ELISA檢測抗血清效價約為1:8000。Western-blot檢測多克隆抗血清可以與Hela和MCF-7細(xì)胞裂解液特異結(jié)合,形成單一的結(jié)合條帶。間接免疫熒光顯示,陽性物質(zhì)分布于Hela和MCF-7細(xì)胞表面。細(xì)胞遷移實驗中抗體封閉組和趨化因子組在細(xì)胞遷移數(shù)量上有明顯的差異。 結(jié)論:成功獲得了純化的GST-SDF-1α融合蛋白和CXCR7多克隆抗體,CXCR7多克隆抗體可抑制SDF-1/CXCR7介導(dǎo)的細(xì)胞遷移。為進(jìn)一步闡明SDF-1/CXCR7在細(xì)胞遷移中的機(jī)制奠定基礎(chǔ)。
[Abstract]:Cell migration is the movement of a cell after it receives a migration signal or feels the concentration gradient of certain substances.Chemokines act as signaling molecules by binding to receptors on the surface of cell membranes to initiate intracellular signals, activate or inhibit the activity of actin binding proteins, and ultimately change the state of the cytoskeleton and cause cell movement.Stromal cell derived factor-1 (SDF-1) is an important chemokine that is widely expressed in a variety of cells and tissues, including immune cells, brain, heart, kidney, liver, lung and spleen, in the immune system.The development of circulatory system and central nervous system plays an important role.It is closely related to HIV infection, inflammation, hematopoietic cell regulation and tumor cell migration.It has been thought that SDF-1 regulates these different processes through CXCR4, the sole receptor.But recently, a new receptor for SDF-1, called CXCR7.CXCR7, was found in many tumor cell lines, activated endothelial cells and fetal liver cells, most of which are not expressed on the surface of normal cells, although these normal cells do not express CXCR7 molecules.However, CXCR7 mRNA was expressed in the cells.Current studies have shown that CXCR7 can promote cell survival in vitro and promote tumor growth in mice. However, the effect of SDF-1/CXCR7 on cell migration is not clear, we have carried out the following two aspects of research:1 prokaryotic expression of SDF-1 偽.The fusion expression vector pGEX-4T-1-SDF-1 偽 was constructed by using the existing pMD-18T-SDF-1 偽 plasmid and pGEX-4T-1 expression vector, and was induced to express in Escherichia coli BL21.After the expression of the target gene was confirmed, the fusion protein was purified by Glutathione Sepharose 4B column, and the antiserum of CXCR7 was prepared, identified and the effect on the migration of MCF-7 cells was studied.The polyclonal antiserum of CXCR7 was prepared by immunizing New Zealand rabbits with two peptides, H458H 459 and KLH crosslinked. The polyclonal antiserum was purified by saturated sulfate-amine method.Elisa was used to detect the titer of antiserum, Western-blot and indirect immunofluorescence to detect the ability of CXCR7 receptor recognition by polyclonal antibodies.Millicell chamber was used to study the effect of SDF-1/CXCR7 on cell migration. The migration cells in antibody blocking group, chemokine group and control group were counted respectively.Results: we successfully constructed the recombinant fusion expression vector pGEX-4T-1-SDF-1 偽 -IPTG to induce the expression of GST-SDF-1 偽, mainly in the form of inclusion body.After washing the inclusion bodies, the purified GST-SDF-1 偽 fusion protein was obtained by using Glutathione Sepharose 4B column.The polyclonal antiserum was obtained by immunizing New Zealand rabbits with the peptide H458H459 linked with KLH. The titer of indirect ELISA detection of polyclonal antiserum was that 1:8000.Western-blot detection of polyclonal antiserum could specifically bind to Hela and MCF-7 cell lysate and form a single binding band.Indirect immunofluorescence showed that the positive substances were distributed on the surface of Hela and MCF-7 cells.In cell migration assay, there were significant differences in the number of cell migration between antibody blocking group and chemokine group.Conclusion: purified GST-SDF-1 偽 fusion protein and CXCR7 polyclonal antibody, CXCR7 polyclonal antibody, can inhibit cell migration mediated by SDF-1/CXCR7.It lays a foundation for further elucidating the mechanism of SDF-1/CXCR7 in cell migration.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 鄭紅,Stephen C Peiper,朱錫華;趨化因子受體CXCR4的結(jié)構(gòu)與功能[J];第三軍醫(yī)大學(xué)學(xué)報;2001年02期

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本文編號：1746498