TNFα和IL-1β靶点抗炎药物筛选细胞模型的构建

发布时间：2018-01-21 04:57

本文关键词： 肿瘤坏死因子α 白介素β 抗炎药物筛选稳定转染　出处：《现代生物医学进展》2015年06期 　论文类型：期刊论文

【摘要】：目的:构建两个高表达人TNFα和IL-1β细胞系,建立抗炎药物筛选细胞模型。方法:运用PCR的方法从载体pCMVSport-TNFα和p CMVSport-IL1β上扩增目的基因,以亚克隆方法将目的基因分别插入真核表达载体pcDNA3.1和pFLAG-CMV中,用单酶切、PCR扩增和基因测序的方法鉴定重组效果,然后将重组成功的质粒转入HEK293细胞系内,挑选能够稳定表达并遗传的单克隆细胞株,用蛋白免疫印迹(Western blot)法分析其表达效果。结果:三种鉴定方法均显示重组质粒构建成功。Western blot结果显示,细胞株T3、T4均能较高表达炎症因子TNF-α;细胞株I2、I3、I5均能较高表达炎症因子IL-1β。结论:成功构建了TNFα和IL-1β靶标的药物筛选细胞模型,为筛选具有抗炎作用的中药提供了一个新平台。
[Abstract]:Objective: to construct two high expression human TNF 伪 and IL-1 尾 cell lines. Methods: PCR method was used to amplify the target gene from vector pCMVSport-TNF 伪 and p CMVSport-IL1 尾. The target gene was inserted into eukaryotic expression vector pcDNA3.1 and pFLAG-CMV by subcloning method. The recombinant effect was evaluated by single enzyme digestion PCR amplification and gene sequencing. Then the recombinant plasmid was transferred into HEK293 cell line to select the stable expression and inheritance of monoclonal cell lines. Results: all the three identification methods showed that the recombinant plasmid was successfully constructed. Western blot showed that the recombinant plasmid was constructed successfully. The expression of inflammatory factor TNF- 伪 was higher in T _ 3 / T _ 4 cell line than in T _ 3 / T _ 4 cell line. Conclusion: the drug screening model of TNF 伪 and IL-1 尾 target was successfully constructed. It provides a new platform for screening Chinese medicine with anti-inflammatory effect.
【作者单位】：北京中医药大学基础医学院;北京中医药大学中药学院;
【基金】：国家自然科学基金项目(81072901) 教育部博士点基金项目(20120013110013) 在读研究生资助项目(2014-JYBZZ-XS-029);在读研究生资助项目(2014-JYBZZ-XS-013)
【分类号】：R329.2
【正文快照】： 前言炎症是指机体对感染、外来物质或其它原因(如抗原抗体复合物)所致损伤的一种反应,炎症的复杂进程既能为机体抵抗外界感染或损伤,也会为宿主带来不同程度的伤害作用。另一方面,炎症反应也可能带来一系列的疾病,最常见的包括类风湿性关节炎,肠道炎症,牛皮癣和多发性硬化症等

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