人黑色素瘤相关抗原MAGE-B18与核因子κB调控蛋白TNIP1相互作用的研究

发布时间：2018-04-17 01:00

本文选题：肿瘤 + 黑色素瘤相关抗原　；参考：《青岛大学》2012年硕士论文

【摘要】：目的：旨在探讨人黑色素瘤相关抗原MAGE-B18与核因子κB调控蛋白TNIP1间的相互作用,同时确定其相互作用的结合位点,从而了解其相互结合的具体分子机制。在明确其分子作用机制后,采用多种手段证实其下游信号转导机制。为该抗原的功能研究奠定基础。方法：将人核因子K B调控蛋白TNIP1及人黑色素瘤相关抗原MAGE-B18共转染293T细胞,使用免疫共沉淀鉴定hMAGE-B18与TNIP1在293T细胞中是否存在相互作用。确定其能够相互结合后,使用PCR方法构建这两种蛋白缺失不同结构域的突变体携带HA及Myc标签的真核表达载体并使其在293T细胞中表达后；再次使用免疫共沉淀确定其具体的结合位点。了解其相互结合的集体分子机制后,使用Western-Blotting等手段观察其下游信号分子的活化情况。并使用流式细胞术观测293T细胞活化后细胞凋亡率的变化。结果：免疫共沉淀结果表明TNIP1与MAGE-B18蛋白在细胞内能够相互结合。成功构建了人核因子K B调控蛋白TNIP1及人黑色素瘤相关抗原MAGE-B18携带有标签蛋白的突变体,并且成功地在293T细胞中进行了表达。并且鉴定出了TNIP1以及MAGE-B18的相互结合位点分别位于311-535aa与114-284aa,使用半定量Western-Blotting验证了TNIP1与MAGE-B18可以发生协同作用,从而影响核因子K B的活化。同时使用流式细胞术证实了TNIP1与MAGE-B18发生协同作用后可以抑制细胞凋亡。结论：黑色素瘤相关抗原MAGE-B18可以与人核因子K B调控蛋白TNIP1在细胞内发生相互作用。同时这两种蛋白可以发生协同效应,从而抑制核因子K B活化。同时,在这两种蛋白的共同作用下可以显著抑制由于NF-κB活化所产生的细胞凋亡这为我们进一步研究MAGE-B18以及TNIP1在肿瘤的发生发展中的作用奠定了基础。
[Abstract]:Objective : To investigate the interaction between human melanoma - associated antigen MAGE - B18 and nuclear factor - 魏B regulatory protein TNIP1 , and to identify the binding sites of its interaction , so as to understand the specific molecular mechanism of the interaction of human melanoma - associated antigen MAGE - B18 .

Methods : Human nuclear factor K - B regulatory protein TNIP1 and human melanoma - associated antigen MAGE - B18 were co - transfected into 293 cells .
The specific binding sites were determined by using immune co - precipitation . The activation of downstream signaling molecules was observed by Western - Blotting and Western - Blotting .

Results : The results showed that TNIP1 and MAGE - B18 protein were able to bind to each other in the cells . The human nuclear factor K - B regulatory protein TNIP1 and human melanoma - associated antigen MAGE - B18 were successfully constructed .

Conclusion : The melanoma - associated antigen MAGE - B18 can interact with the human nuclear factor K - B regulatory protein TNIP1 in the cell . At the same time , the two proteins can inhibit the activation of nuclear factor K B . At the same time , the apoptosis induced by NF - 魏B activation can be significantly inhibited under the coaction of these two proteins , which lays a foundation for further study on the role of MAGE - B18 and TNIP1 in the development of tumor .

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5

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