应用RNA干扰技术研究线虫酪氨酸磷酸酶PTP-2的生物学功能
发布时间：2018-07-26 13:01
【摘要】： SHP-2是一种存在于哺乳动物体内的非跨膜型蛋白酪氨酸磷酸酶，它在调节细胞生物的多种信号转导过程及控制细胞活性中发挥着重要的作用。为研究SHP-2的生物学功能，我们选择线虫(Caenorhabditis elegans)作为模式生物，，应用RNA干扰技术对PTP-2(线虫中SHP-2)的功能进行了研究。首先，我们设计并合成引物，利用PCR方法从线虫的cDNA文库中扩增出ptp-2基因，克隆到表达载体pGex-2T中，并在E-coli BL21codonplus pLysS中得到了高效表达；其次，我们应用制备电泳获得了高纯度的GST-PTP-2融合蛋白，并将得到的融合蛋白免疫家兔，得到了有较高特异性的PTP-2多克隆抗体；最后，在上述工作基础上，我们应用RNAi技术干扰ptp-2基因在野生型线虫中的表达，通过对线虫生物学表型的研究，我们发现干扰组线虫在寿命以及非正常死亡方面均与对照组线虫有明显差别，说明PTP-2在调控线虫阴道肌肉系统发育的过程中发挥了重要的作用，同时也参与寿命相关的信号传导通路的调控。
[Abstract]:SHP-2 is a non-transmembrane protein tyrosine phosphatase which exists in mammalian. It plays an important role in regulating many signal transduction processes and controlling cell activity. In order to study the biological function of SHP-2, (Caenorhabditis elegans) was selected as the model organism, and the function of PTP-2 (SHP-2 in nematode) was studied by RNA interference technique. Firstly, we designed and synthesized primers, amplified ptp-2 gene from cDNA library of nematode by PCR method, cloned it into expression vector pGex-2T, and obtained high expression in E-coli BL21codonplus pLysS. High purity GST-PTP-2 fusion protein was obtained by using preparation electrophoresis, and the fusion protein was immunized to rabbits, and a highly specific PTP-2 polyclonal antibody was obtained. Finally, on the basis of the above work, We used RNAi technique to interfere with the expression of ptp-2 gene in wild nematode. By studying the biological phenotype of nematode, we found that there were significant differences in longevity and abnormal death between the interference group and the control group. It is suggested that PTP-2 plays an important role in regulating the development of vagina muscle system of nematodes, and it is also involved in the regulation of signal transduction pathway related to longevity.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
本文编号：2146093
[Abstract]:SHP-2 is a non-transmembrane protein tyrosine phosphatase which exists in mammalian. It plays an important role in regulating many signal transduction processes and controlling cell activity. In order to study the biological function of SHP-2, (Caenorhabditis elegans) was selected as the model organism, and the function of PTP-2 (SHP-2 in nematode) was studied by RNA interference technique. Firstly, we designed and synthesized primers, amplified ptp-2 gene from cDNA library of nematode by PCR method, cloned it into expression vector pGex-2T, and obtained high expression in E-coli BL21codonplus pLysS. High purity GST-PTP-2 fusion protein was obtained by using preparation electrophoresis, and the fusion protein was immunized to rabbits, and a highly specific PTP-2 polyclonal antibody was obtained. Finally, on the basis of the above work, We used RNAi technique to interfere with the expression of ptp-2 gene in wild nematode. By studying the biological phenotype of nematode, we found that there were significant differences in longevity and abnormal death between the interference group and the control group. It is suggested that PTP-2 plays an important role in regulating the development of vagina muscle system of nematodes, and it is also involved in the regulation of signal transduction pathway related to longevity.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
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