单纯疱疹病毒1型ICP0真核载体的构建及其对巨噬细胞分泌活性的影响

发布时间：2018-02-03 07:23

本文关键词： 单纯疮疹病毒1型 GFP-ICP0 巨噬细胞细胞因子 NO　出处：《南华大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：构建ICP0真核表达载体pEGFP／ICP0，转染巨噬细胞，检测ICP0在巨噬细胞中的表达与定位。研究GFP-ICP0融合蛋白瞬时高表达对激活巨噬细胞分泌IL-1β、TNF-α以及NO的影响，为进一步探讨ICP0的致病性提供一定的实验依据。方法：限制性内切酶双酶切质粒PT7-110，将目的基因连接至真核表达载体pEGFP—C1，构建ICP0真核表达载体pEGFP／ICP0。转染至巨噬细胞后，荧光显微镜观察ICP0在巨噬细胞中的定位，western blotting检测ICP0蛋白的表达。用TNF-α和IL-1β定量试剂盒检测LPS激活的巨噬细胞分泌TNF-α和IL-1β的水平，用Griess试剂测定经刺激后的小鼠巨噬细胞产生的NO水平。结果：限制性内切酶双酶切质粒PT7-110，将该目的片断亚克隆至真核载体pEGFP-C1上，所构建的真核表达载体pEGFP／ICP0转染巨噬细胞，荧光显微镜观察GFP-ICP0融合蛋白定位于细胞核，western blotting结果表明pEGFP／ICP0可以在真核细胞中表达大小约为107KD的GFP-ICP0融合蛋白。转染24h后的细胞加入终浓度为100ng／mL的LPS诱导，分别收集诱导后12h、24h、48h细胞培养上清，ELISA法检测上清中细胞因子TNF-α、IL-1β含量的变化，Griess试剂测定上清液中的NO水平。结果表明，GFP-ICP0融合蛋白瞬时表达可上调LPS诱导的巨噬细胞细胞因子TNF-α、IL-1β以及NO的分泌，与各对照组比较，结果有显著性差异(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- 伪 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- 伪 and IL-1 尾 by LPS activated macrophages were detected by TNF- 伪 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- 伪 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- 伪 induced by LPS. The levels of IL-1 尾 and no were significantly different from those of the control group (P < 0.05).
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373

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