【摘要】：目的DAPK3是本研究組于2011年利用酵母雙雜交系統(tǒng)篩選出來的LOH12CR1可能的直接互作蛋白。LOH12CR1可能是—抑癌基因,但其具體生物學(xué)功能及作用機制尚不清楚。驗證LOH12CR1和DAPK3在細(xì)胞內(nèi)是否真正存在互作關(guān)系,為LOH12CR1功能的研究提供切入點。 方法 1.構(gòu)建pCDNA3.1-myc-his-LOH12CR1、 pCMV-tag2-DAPK3重組載體,并對HEK293細(xì)胞共轉(zhuǎn)染LOH12CR1和DAPK3重組載體,利用免疫共沉淀技術(shù)驗證兩者在細(xì)胞內(nèi)的的相互作用； 2.將LOH12CR1和DAPK3共轉(zhuǎn)染于HEK293細(xì)胞,通過激光共聚焦技術(shù)觀察兩者在HEK293內(nèi)的免疫熒光亞細(xì)胞水平共定位情況。 結(jié)果 1.免疫共沉淀實驗顯示LOH12CR1與DAPK3在HEK293細(xì)胞內(nèi)存在相互作用； 2.免疫熒光亞細(xì)胞共定位實驗初步提示LOH12CR1和DAPK3存在亞細(xì)胞水平共定位,且主要共定位于胞漿。 結(jié)論本研究證實了LOH12CR1與DAPK3在細(xì)胞內(nèi)的相互作用關(guān)系,目前已知DAPK3的主要生物學(xué)功能為促進(jìn)凋亡,提示LOH12CR1可能通過與DAPK3的相互作用參與調(diào)控細(xì)胞凋亡過程,其具體作用機制我們將進(jìn)行進(jìn)一步的研究以明確。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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