IFN-α刺激相关细胞蛋白TIIMPSC和S24v2的功能分析
发布时间：2018-12-24 11:08
【摘要】： 干扰素具有广泛的生物学功能，如抗病毒、抗肿瘤、免疫调节、调控细胞增殖和分化等。IFN-α作为Ⅰ型干扰素家族的重要成员，主要产生于非免疫细胞，其功能的多样性是通过诱导细胞表达多种蛋白质而实现的。IFN-α作用于细胞可启动一条或多条不同的信号转导途径，不同的信号途径又可转录出不同的IFN刺激基因。针对其调节非免疫细胞发动特定效应抵抗病毒的特点，探索非免疫细胞对IFN-α刺激反应的机理，和由此机理所导致的各种可能的生物学意义具有很大的意义。 本论文涉及IFN-α刺激人成纤维细胞KMB-17株的所引起的细胞内基因表达的变化，通过IFN-α刺激人二倍体细胞KMB-17后的基因反应的差异分析，成功克隆到两个差异基因，，其中IFN-α刺激特异表达得蛋白分子为类CD69Ⅱ型膜蛋白(typeⅡmembrane protein similar to CD69，TⅡMPSC)TⅡMPSC，长度为697bp的cDNA基因(基因库号码：AB015628)；另一个差异蛋白为IFN-α刺激下调表达蛋白，cds长度为593bp(基因库号码：NM_001026)，经对比分析确定为核糖体复合物亚单位蛋白S24变异体2(ribosomal protein S24 transcript variant 2，S24v2)。我们分别将基因克隆到融合表达载体pET-28a和pGEX-5x-1，构建了pET-TⅡMPSC和pGEX-S24v2表达质粒，表达纯化获得较高纯度的目的蛋白，用该蛋白免疫小鼠后制备的特异抗体，蛋白印迹实验表明原核表达的的蛋白能被该抗体特异识别。 对TⅡMPSCcDNA编码蛋白的分析表明，该蛋白具有膜蛋白特性，这一点为该蛋白在人成纤维细胞包膜的免疫荧光亚细胞定位所证实。细胞增殖实验和JAK／STAT信号通路相关研究结果提示IFN-α刺激细胞后上调表达的TⅡMPSC蛋白能够明显推进人成纤维细胞进入S期，其在JAK／STAT信号通路中所处的位置和功能分析提供了一定的线索：免疫共沉淀实验实验显示了TⅡMPSC蛋白通过JAK／STAT信号传导介导的信号转导和转录激活。 S24v2作为胞内核糖体复合物的亚单位之一正常情况下参与了核糖体构成和蛋白翻译的过程，IFN-α的刺激能够特异阻滞该蛋白的表达，在转染pcDNA-S24v2的经IFN-α预处理的KMB-17细胞中，脊髓灰质炎Ⅰ型病毒(Polio virus，typeⅠ)的增殖得到了补偿。通过检测在转染pcDNA-S24v2的KMB-17细胞中的病毒滴度，下调或干扰S24v2的表达可能延迟病毒蛋白的合成，从而影响其复制，可以推测S24v2对于病毒复制及病毒蛋白的合成均具有重要作用。
[Abstract]:Interferon has a wide range of biological functions, such as anti-virus, anti-tumor, immunomodulation, regulating cell proliferation and differentiation, etc. As an important member of interferon type I family, IFN- 伪 is mainly produced in non-immune cells. Its function diversity is realized by inducing cells to express many kinds of proteins. IFN- 伪 can activate one or more different signal transduction pathways, and different signal pathways can transcribe different IFN stimulating genes. In view of its characteristics of regulating non-immune cells to initiate specific effects against virus, it is of great significance to explore the mechanism of non-immune cells' response to IFN- 伪 stimulation and the possible biological significance resulting from this mechanism. This paper deals with the changes of gene expression induced by IFN- 伪 stimulation of human fibroblast KMB-17 strain. Two differentially expressed genes were successfully cloned by differential analysis of the gene response of IFN- 伪 to human diploid cell KMB-17. Among them, IFN- 伪 stimulated the specific expression of CD69 鈪
本文编号：2390544
[Abstract]:Interferon has a wide range of biological functions, such as anti-virus, anti-tumor, immunomodulation, regulating cell proliferation and differentiation, etc. As an important member of interferon type I family, IFN- 伪 is mainly produced in non-immune cells. Its function diversity is realized by inducing cells to express many kinds of proteins. IFN- 伪 can activate one or more different signal transduction pathways, and different signal pathways can transcribe different IFN stimulating genes. In view of its characteristics of regulating non-immune cells to initiate specific effects against virus, it is of great significance to explore the mechanism of non-immune cells' response to IFN- 伪 stimulation and the possible biological significance resulting from this mechanism. This paper deals with the changes of gene expression induced by IFN- 伪 stimulation of human fibroblast KMB-17 strain. Two differentially expressed genes were successfully cloned by differential analysis of the gene response of IFN- 伪 to human diploid cell KMB-17. Among them, IFN- 伪 stimulated the specific expression of CD69 鈪
本文编号：2390544
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