LOH12CR1互作蛋白DAPK3的鉴定
发布时间：2019-04-18 13:47
【摘要】：目的DAPK3是本研究组于2011年利用酵母双杂交系统筛选出来的LOH12CR1可能的直接互作蛋白。LOH12CR1可能是—抑癌基因,但其具体生物学功能及作用机制尚不清楚。验证LOH12CR1和DAPK3在细胞内是否真正存在互作关系,为LOH12CR1功能的研究提供切入点。 方法 1.构建pCDNA3.1-myc-his-LOH12CR1、 pCMV-tag2-DAPK3重组载体,并对HEK293细胞共转染LOH12CR1和DAPK3重组载体,利用免疫共沉淀技术验证两者在细胞内的的相互作用； 2.将LOH12CR1和DAPK3共转染于HEK293细胞,通过激光共聚焦技术观察两者在HEK293内的免疫荧光亚细胞水平共定位情况。 结果 1.免疫共沉淀实验显示LOH12CR1与DAPK3在HEK293细胞内存在相互作用； 2.免疫荧光亚细胞共定位实验初步提示LOH12CR1和DAPK3存在亚细胞水平共定位,且主要共定位于胞浆。 结论本研究证实了LOH12CR1与DAPK3在细胞内的相互作用关系,目前已知DAPK3的主要生物学功能为促进凋亡,提示LOH12CR1可能通过与DAPK3的相互作用参与调控细胞凋亡过程,其具体作用机制我们将进行进一步的研究以明确。
[Abstract]:Aim DAPK3 is a possible direct interacting protein of LOH12CR1 screened by yeast two hybrid system in 2011. LOH12CR1 may be a tumor suppressor gene, but the biological function and mechanism of LOH12CR1 are still unclear. To verify whether there is a real interaction between LOH12CR1 and DAPK3 in cells and to provide a starting point for the study of LOH12CR1 function. Method 1. To construct the recombinant vector of pCDNA3.1-myc-his-LOH12CR1, pCMV-tag2-DAPK3 and co-transfect the recombinant vector of LOH12CR1 and DAPK3 into HEK293 cells, and to verify the interaction between them by immunoprecipitation technique. LOH12CR1 and DAPK3 were co-transfected into HEK293 cells. Co-localization of the two cells in HEK293 was observed by laser confocal microscopy. Outcome 1. Immunoprecipitation assay showed that there was interaction between LOH12CR1 and DAPK3 in HEK293 cells. Immunofluorescence subcellular co-localization assay suggested that LOH12CR1 and DAPK3 were co-located at the subcellular level and mainly co-located in cytoplasm. Conclusion this study confirmed the interaction between LOH12CR1 and DAPK3 in cells. The main biological function of DAPK3 is known to promote apoptosis, suggesting that LOH12CR1 may be involved in the regulation of apoptosis through the interaction with DAPK3. The specific mechanism of its action will be further studied to clarify.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2460086
[Abstract]:Aim DAPK3 is a possible direct interacting protein of LOH12CR1 screened by yeast two hybrid system in 2011. LOH12CR1 may be a tumor suppressor gene, but the biological function and mechanism of LOH12CR1 are still unclear. To verify whether there is a real interaction between LOH12CR1 and DAPK3 in cells and to provide a starting point for the study of LOH12CR1 function. Method 1. To construct the recombinant vector of pCDNA3.1-myc-his-LOH12CR1, pCMV-tag2-DAPK3 and co-transfect the recombinant vector of LOH12CR1 and DAPK3 into HEK293 cells, and to verify the interaction between them by immunoprecipitation technique. LOH12CR1 and DAPK3 were co-transfected into HEK293 cells. Co-localization of the two cells in HEK293 was observed by laser confocal microscopy. Outcome 1. Immunoprecipitation assay showed that there was interaction between LOH12CR1 and DAPK3 in HEK293 cells. Immunofluorescence subcellular co-localization assay suggested that LOH12CR1 and DAPK3 were co-located at the subcellular level and mainly co-located in cytoplasm. Conclusion this study confirmed the interaction between LOH12CR1 and DAPK3 in cells. The main biological function of DAPK3 is known to promote apoptosis, suggesting that LOH12CR1 may be involved in the regulation of apoptosis through the interaction with DAPK3. The specific mechanism of its action will be further studied to clarify.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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