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  本文關(guān)鍵詞： 單純瘡疹病毒1型 GFP-ICP0 巨噬細(xì)胞 細(xì)胞因子 NO　出處：《南華大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：構(gòu)建ICP0真核表達(dá)載體pEGFP／ICP0，轉(zhuǎn)染巨噬細(xì)胞，檢測ICP0在巨噬細(xì)胞中的表達(dá)與定位。研究GFP-ICP0融合蛋白瞬時高表達(dá)對激活巨噬細(xì)胞分泌IL-1β、TNF-α以及NO的影響，為進(jìn)一步探討ICP0的致病性提供一定的實(shí)驗(yàn)依據(jù)。 方法：限制性內(nèi)切酶雙酶切質(zhì)粒PT7-110，將目的基因連接至真核表達(dá)載體pEGFP—C1，構(gòu)建ICP0真核表達(dá)載體pEGFP／ICP0。轉(zhuǎn)染至巨噬細(xì)胞后，熒光顯微鏡觀察ICP0在巨噬細(xì)胞中的定位，western blotting檢測ICP0蛋白的表達(dá)。用TNF-α和IL-1β定量試劑盒檢測LPS激活的巨噬細(xì)胞分泌TNF-α和IL-1β的水平，用Griess試劑測定經(jīng)刺激后的小鼠巨噬細(xì)胞產(chǎn)生的NO水平。 結(jié)果：限制性內(nèi)切酶雙酶切質(zhì)粒PT7-110，將該目的片斷亞克隆至真核載體pEGFP-C1上，所構(gòu)建的真核表達(dá)載體pEGFP／ICP0轉(zhuǎn)染巨噬細(xì)胞，熒光顯微鏡觀察GFP-ICP0融合蛋白定位于細(xì)胞核，western blotting結(jié)果表明pEGFP／ICP0可以在真核細(xì)胞中表達(dá)大小約為107KD的GFP-ICP0融合蛋白。轉(zhuǎn)染24h后的細(xì)胞加入終濃度為100ng／mL的LPS誘導(dǎo)，分別收集誘導(dǎo)后12h、24h、48h細(xì)胞培養(yǎng)上清，ELISA法檢測上清中細(xì)胞因子TNF-α、IL-1β含量的變化，Griess試劑測定上清液中的NO水平。結(jié)果表明，GFP-ICP0融合蛋白瞬時表達(dá)可上調(diào)LPS誘導(dǎo)的巨噬細(xì)胞細(xì)胞因子TNF-α、IL-1β以及NO的分泌，與各對照組比較，結(jié)果有顯著性差異(采用方差分析，，P＜0.05)。
[Abstract]:Objective: to construct ICP0 eukaryotic expression vector pEGFP / ICP0 and transfect it into macrophages. To detect the expression and localization of ICP0 in macrophages, and to study the effect of transient overexpression of GFP-ICP0 fusion protein on the secretion of IL-1 尾 -TNF- 偽 and no by macrophages. To further explore the pathogenicity of ICP0 to provide a certain experimental basis. Methods: the plasmid PT7-110 was digested by restriction endonuclease and the target gene was ligated to eukaryotic expression vector pEGFP-C1. ICP0 eukaryotic expression vector pEGFP / ICP0 was constructed. After transfection into macrophages, the localization of ICP0 in macrophages was observed by fluorescence microscope. Western. The expression of ICP0 protein was detected by blotting and the levels of TNF- 偽 and IL-1 尾 by LPS activated macrophages were detected by TNF- 偽 and IL-1 尾 quantitative kit. The level of no produced by stimulated mouse macrophages was determined by Griess reagent. Results: restriction endonuclease digestion plasmid PT7-110 was subcloned into eukaryotic vector pEGFP-C1. The constructed eukaryotic expression vector pEGFP/ICP0 was transfected into macrophages and the GFP-ICP0 fusion protein was observed to be located in the nucleus by fluorescence microscope. Western. Blotting results showed that pEGFP/ICP0 could express a GFP-ICP0 fusion protein of about 107 KD in eukaryotic cells. After 24 hours of transfection, the final concentration of GFP-ICP0 fusion protein was as follows: 1. 100 ng / mL LPS was induced. The levels of cytokine TNF- 偽 and IL-1 尾 in the supernatant were detected by Elisa. The level of no in supernatant was determined by Griess reagent. The results showed that the transient expression of GFP-ICP0 fusion protein could up-regulate the macrophage cytokine TNF- 偽 induced by LPS. The levels of IL-1 尾 and no were significantly different from those of the control group (P < 0.05).
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊曉儀,林鍵,吳文言;重組蛋白包涵體的復(fù)性研究[J];生命科學(xué)研究;2004年02期

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