HBx编码蛋白质通过调节SPRY1编码蛋白质激活MAPK信号转导通研究
发布时间：2018-08-13 14:34
【摘要】： 乙型肝炎病毒(HBV)基因组中的X基因及其编码产物X蛋白(hepatitis B virus X protein, HBx)在HBV的复制中起着重要的调节作用。HBx参与多条细胞信号转导通路,对细胞的增殖,凋亡发挥重要的调节作用。它主要与细胞信号通路中一些关键性的调节蛋白质协同完成对细胞信号转导通路的调节作用。这些调控作用包括激活宿主细胞和病毒自身基因的转录、调控凋亡、抑制细胞中受损DNA的外切修复反应以及激活细胞中的信号转导通路如有丝分裂原(MAPK)、JAK、STAT的级联反应等。但是在HBx参与众多信号转导通路中,我们关注HBx激活MAPK信号转导通路,该通路是一条高度保守的模块,与细胞各种功能有关,包括细胞增殖,分化和迁移等,并且在肿瘤形成和迁移过程中发挥巨大的调节作用。但是目前并未发现HBx直接作用与MAPK信号中的蛋白酶受体,其激活的潜在分子机制尚未阐明。通过本课题的研究来初步阐明HBx编码的蛋白质在肝脏中是通过调节MAPK的抑制受体---SPRY1基因编码的蛋白质来激活MAPK信号通路。为了阐明该机制,本课题采取了一系列实验研究：(1)构建Flag-HBx、Myc-SPRY1 Myc-SPRY1-53-Mut真核表达载体；(2)确定Flag-HBx和Myc-SPRY1蛋白质之间是否存在相互作用；(3)探索Flag-HBx编码的蛋白质与Myc-SPRY1编码蛋白质相互作用结构域的研究；(4)Myc-SPRY1编码蛋白质是否影响Flag-HBx在哺乳动物HEK293细胞中的蛋白质表达水平；(5)AP-1荧光素酶报告基因实验研究Flag-HBx和Myc-SPRY1编码的蛋白质对AP-1转录因子的影响；(6)研究Flag-HBx和Myc-SPRY1编码的蛋白质对HEK293细胞凋亡、增殖的功能影响。所得结论如下： 1.成功构建Flag-HBx、Myc-SPRY1、Myc-SPRY1-53-Mut,真核表达载体； 2.通过免疫共沉淀(Co-IP)和Pull-down实验确定了Flag-HBx和Myc-SPRY1在哺乳动物HEK293细胞体内外存在相互作用； 3.通过免疫共沉淀实验表明SPRY1第53位酪氨酸突变体并不影响它们之间的相互作用,但是AP-1荧光素酶报告基因实验表明SPRY1第53位酪氨酸突变体影响它与HBx之间对细胞信号转导通路的激活作用,因此我们推测它们相互作用区域是包括这一点的一段序列； 4. Myc-SPRY1编码蛋白质影响Flag-HBx编码的蛋白质在哺乳动物HEK293细胞的表达水平,并且Flag-HBx编码的蛋白质水平随着Myc-SPRY1编码蛋白质表达水平的增加而增加； 5. Flag-HBx协同Myc-SPRY1对核转录因子AP-1-luc的转录活性具有激活效应； 6.通过细胞凋亡实验和细胞增殖实验发现,Flag-HBx促进HEK293细胞的增殖,抑制HEK293细胞的凋亡；并且发现当共转染Myc-SPRY1和Flag-HBx到HEK293细胞中时,Myc-SPRY1增加了Flag-HBx对HEK293细胞的抗凋亡作用,并且Myc-SPRY1上调了Flag-HBx对HEK293细胞的增殖水平的促进作用。
[Abstract]:X gene and X protein (hepatitis B virus X protein, HBx) in the genome of hepatitis B virus (HBV) play an important role in the replication of HBV. HBX participates in many cell signal transduction pathways and proliferates cells. Apoptosis plays an important regulatory role. It mainly works with some key regulatory proteins in cell signaling pathway to regulate cell signal transduction pathway. These effects include activation of transcription of host cells and virus genes, regulation of apoptosis, inhibition of extracellular repair of damaged DNA in cells and activation of signal transduction pathways in cells, such as the cascade of mitogen (MAPK) (MAPK) JAKTSTAT, and so on. However, among the many signal transduction pathways in which HBx is involved, we focus on the activation of MAPK signal transduction pathway by HBx, which is a highly conserved module related to various cellular functions, including cell proliferation, differentiation and migration. And play a huge regulatory role in tumor formation and migration. However, no direct interaction of HBx with protease receptor in MAPK signal has been found, and the underlying molecular mechanism of its activation has not been elucidated. In this study, we preliminarily demonstrated that the HBx encoded protein activates the MAPK signaling pathway by regulating the protein encoded by the suppressor SPRY1 gene of MAPK in the liver. In order to elucidate the mechanism, a series of experimental studies were carried out: (1) constructing the eukaryotic expression vector of Flag-HBxSPRY1 Myc-SPRY1-53-Mut, (2) determining the interaction between Flag-HBx and Myc-SPRY1 protein; (3) to explore the interaction domain between Flag-HBx encoded proteins and Myc-SPRY1 encoded proteins, (4) whether Myc-SPRY1 encoded proteins affect the expression of Flag-HBx in mammalian HEK293 cells; (5) the effects of Flag-HBx and Myc-SPRY1 encoded proteins on AP-1 transcription factors were studied by AP-1 luciferase reporter gene experiment. (6) the effects of Flag-HBx and Myc-SPRY1 encoded proteins on the apoptosis and proliferation of HEK293 cells were studied. The conclusions are as follows: 1. The eukaryotic expression vector of Flag-HBxCPRY1, Myc-SPRY1-53-Mut. was successfully constructed. The interaction between Flag-HBx and Myc-SPRY1 in mammalian HEK293 cells in vivo and in vitro was confirmed by immunoprecipitation (Co-IP) and Pull-down assay. The results of immunoprecipitation showed that tyrosine mutants at position 53 of SPRY1 did not affect the interaction between them. However, the AP-1 luciferase reporter gene experiment showed that tyrosine mutants at position 53 of SPRY1 affect the activation of signal transduction pathway between SPRY1 and HBx, so we speculate that their interaction region is a sequence including this. 4. Myc-SPRY1 encoded protein affects the expression level of Flag-HBx encoded protein in mammalian HEK293 cells, and Flag-HBx protein level increases with the increase of Myc-SPRY1 protein expression level. 5. Flag-HBx combined with Myc-SPRY1 could activate the transcription activity of nuclear transcription factor AP-1-luc. It was found that Flag-HBx promoted the proliferation of HEK293 cells and inhibited the apoptosis of HEK293 cells, and that Myc-SPRY1 increased the anti-apoptotic effect of Flag-HBx on HEK293 cells when co-transfected with Myc-SPRY1 and Flag-HBx into HEK293 cells. Myc-SPRY1 upregulated the promotion of Flag-HBx on the proliferation of HEK293 cells.
【学位授予单位】：兰州理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373
本文编号：2181272
[Abstract]:X gene and X protein (hepatitis B virus X protein, HBx) in the genome of hepatitis B virus (HBV) play an important role in the replication of HBV. HBX participates in many cell signal transduction pathways and proliferates cells. Apoptosis plays an important regulatory role. It mainly works with some key regulatory proteins in cell signaling pathway to regulate cell signal transduction pathway. These effects include activation of transcription of host cells and virus genes, regulation of apoptosis, inhibition of extracellular repair of damaged DNA in cells and activation of signal transduction pathways in cells, such as the cascade of mitogen (MAPK) (MAPK) JAKTSTAT, and so on. However, among the many signal transduction pathways in which HBx is involved, we focus on the activation of MAPK signal transduction pathway by HBx, which is a highly conserved module related to various cellular functions, including cell proliferation, differentiation and migration. And play a huge regulatory role in tumor formation and migration. However, no direct interaction of HBx with protease receptor in MAPK signal has been found, and the underlying molecular mechanism of its activation has not been elucidated. In this study, we preliminarily demonstrated that the HBx encoded protein activates the MAPK signaling pathway by regulating the protein encoded by the suppressor SPRY1 gene of MAPK in the liver. In order to elucidate the mechanism, a series of experimental studies were carried out: (1) constructing the eukaryotic expression vector of Flag-HBxSPRY1 Myc-SPRY1-53-Mut, (2) determining the interaction between Flag-HBx and Myc-SPRY1 protein; (3) to explore the interaction domain between Flag-HBx encoded proteins and Myc-SPRY1 encoded proteins, (4) whether Myc-SPRY1 encoded proteins affect the expression of Flag-HBx in mammalian HEK293 cells; (5) the effects of Flag-HBx and Myc-SPRY1 encoded proteins on AP-1 transcription factors were studied by AP-1 luciferase reporter gene experiment. (6) the effects of Flag-HBx and Myc-SPRY1 encoded proteins on the apoptosis and proliferation of HEK293 cells were studied. The conclusions are as follows: 1. The eukaryotic expression vector of Flag-HBxCPRY1, Myc-SPRY1-53-Mut. was successfully constructed. The interaction between Flag-HBx and Myc-SPRY1 in mammalian HEK293 cells in vivo and in vitro was confirmed by immunoprecipitation (Co-IP) and Pull-down assay. The results of immunoprecipitation showed that tyrosine mutants at position 53 of SPRY1 did not affect the interaction between them. However, the AP-1 luciferase reporter gene experiment showed that tyrosine mutants at position 53 of SPRY1 affect the activation of signal transduction pathway between SPRY1 and HBx, so we speculate that their interaction region is a sequence including this. 4. Myc-SPRY1 encoded protein affects the expression level of Flag-HBx encoded protein in mammalian HEK293 cells, and Flag-HBx protein level increases with the increase of Myc-SPRY1 protein expression level. 5. Flag-HBx combined with Myc-SPRY1 could activate the transcription activity of nuclear transcription factor AP-1-luc. It was found that Flag-HBx promoted the proliferation of HEK293 cells and inhibited the apoptosis of HEK293 cells, and that Myc-SPRY1 increased the anti-apoptotic effect of Flag-HBx on HEK293 cells when co-transfected with Myc-SPRY1 and Flag-HBx into HEK293 cells. Myc-SPRY1 upregulated the promotion of Flag-HBx on the proliferation of HEK293 cells.
【学位授予单位】：兰州理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373
【参考文献】
相关期刊论文 前3条
1 孙玮;胡和平;;HBx的研究进展[J];山东医药;2007年27期
2 汪琼;董俊兴;;乙型肝炎病毒X基因及HBx蛋白的研究进展[J];生物技术通讯;2006年01期
3 朱明华,戴益民,詹甸洲;HBxAg增加p53蛋白在肝癌细胞内积聚[J];中华病理学杂志;1999年01期
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