人类乙型肝炎病毒前-S1蛋白反式调节新基因PS1TP5的克隆表达与部分功能研究

发布时间：2018-06-24 03:45

本文选题：乙型肝炎病毒 + 前-S1蛋白　；参考：《山西医科大学》2007年博士论文

【摘要】： 乙型肝炎病毒的持续感染至今仍然是一个全球性的健康问题,不仅引起急慢性病毒性肝炎,而且与肝纤维化、肝细胞癌的发生发展密切相关。然而对于HBV感染目前尚无特效的治疗方法,这主要是由于HBV感染引起病毒性肝炎的发病机制尚不清楚。目前认为,HBV进入肝细胞后,病毒基因组及其编码的蛋白与肝细胞基因及蛋白之间的相互作用是决定HBV复制、表达、免疫逃逸、慢性感染、肝纤维化及致恶性转化的关键因素。特别是近年来随着对HBV致病机制研究的深入,发现在HBV与肝细胞相互作用的过程中涉及到复杂的反式调节机制,肝炎病毒蛋白在肝细胞中的表达对于肝细胞的基因表达谱产生影响,这一作用可能与其致病机制有关。因此,寻找肝细胞中与HBV各抗原成分反式调节的相关基因,并进一步研究它的作用机制、作用效应及相互影响,对明确HBV的致病机制,寻找有效防治方法具有重要意义。对于筛选得到的未知功能基因的克隆表达与功能研究是病毒性肝炎发病机制研究领域中创新知识的重要源泉,同时也是人类基因组计划和后基因组计划的重要内容。本研究利用转染了HBV前-S1蛋白基因片段表达载体的肝细胞和仅转染空载体的肝细胞为材料,提取其mRNA应用SSH技术研究了HBV前-S1蛋白的反式调节靶基因,筛选得到一未知功能基因,经斑点杂交技术验证后,确定其基因编码序列,命名为乙型肝炎病毒前-S1蛋白反式调节蛋白5(PS1TP5),GenBankAccession:AY427953。发现一种新基因,将其编码的新蛋白结构与生物学功能,与生物学和临床医学之间的相互关系,以及表达调控机制阐明,是目前分子生物学研究领域中最具有挑战性的工作。为此本研究做了以下四方面工作。 1.提取HepG2细胞mRNA,利用RT-PCR方法在PS1TP5基因的两端分别引入EcoRI和BglⅡ酶切位点,获得了438bp的目的基因片段,连接至pGEM-T载体,测序正确后插入至原核表达载体pET-32a(+)中,转化BL21宿主菌,1 mmol/LIPTG在30℃诱导2h、3h、4h后均获得了带有组氨酸标签的重组融合蛋白的表达。 2.利用ExPASy proteomics server对PS1TP5进行蛋白理化性质与结构功能预测分析。PS1TP5相对分子量为15853.4,理论pI为9.41,不同条件下的消光系数为17335 M~(-1)cm~(-1)或16960 M~(-1)cm~(-1)(280nm),在体外哺乳动物网状细胞中的半寿期为30hr,并且无卷曲螺旋区域,但具有4个疏水区;无特殊二级结构,有3个蛋白激酶磷酸化位点,不具有跨膜螺旋结构,无信号肽,定位于细胞浆中。 3.将引入EcoRI和BglⅡ酶切位点的PS1TP5基因片段插入至酵母细胞表达载体pGBKT7中,转化AH109酵母菌株并获得表达,与含有白细胞文库质粒的Y187酵母菌株配合,进行酵母双杂交筛选获得了与PS1TP5具有相互作用的已知功能蛋白23种,包括白细胞黏附蛋白p150,95、白介素2受体γ链、PALM2-AKAP2蛋白、真核翻译起始因子4A、β2微球蛋白、钠-氢交换溶质载体家族9、无唾液酸糖蛋白受体1(ASGR1)、钙网织蛋白、MHC类Ⅱ型淋巴细胞抗原、细胞色素C氧化酶亚型1、淋巴细胞抗原86(LY86)、淋巴细胞溶质蛋白1等。结果提示PS1TP5可能参与肝细胞增殖分化、信息传递以及生长代谢。此外还筛选得到一个新基因,命名为乙型肝炎病毒前-S1蛋白反式激活蛋白5结合蛋白1(PS1TP5BP1),GenBank accession为DQ471327。 4.构建pcDNA3.1/myc-His(-)A-PS1TP5真核表达重组质粒,与空质粒分别转染HepG2细胞,两者互为平行对照,提取转染后的细胞mRNA,成功地构建了PS1TP5蛋白反式激活相关基因差异表达的cDNA消减文库,筛选获得了23条已知的反式上调基因,包括信号转导分子:跨膜蛋白家族4超家族成员1(TM4SF1)、溶质载体家族7成员5(SLC7A5)、磷酸肌醇酶2(IMPA2)、信号序列受体-β、环氧化物酶1、蛋白激酶BRPK等;蛋白因子:核糖体蛋白S16、核糖体蛋白LP0、核糖体蛋白LP2、核糖体蛋白S7、核糖体蛋白L31、丛生蛋白转录突变体1等;转录因子:真核延伸因子1;能量代谢有关的酶:NADH脱氢酶1、肝丙酮酸脱氢酶、细胞色素C氧化酶亚单位8A、烯醇酶1、腺苷脱氨酶等。结果提示PS1TP5可能参与人类免疫调节、物质代谢、信号转导。特别是与肝纤维化形成、肿瘤发生发展有密切关系。此外还筛选得到一个新基因,命名为乙型肝炎病毒前-S1蛋白反式激活蛋白5反式激活蛋白1(PS1TP5TP1),GenBankaccession为DQ487761。
[Abstract]:The continuous infection of hepatitis B virus is still a global health problem, which not only causes acute and chronic viral hepatitis, but also is closely related to the development of liver fibrosis and hepatocellular carcinoma. However, there is no special treatment for HBV infection, which is mainly due to the pathogenesis of viral hepatitis caused by HBV infection. It is not clear that the interaction between the virus genome and its encoded proteins and the gene and protein of hepatocytes is the key factor in determining HBV replication, expression, immune escape, chronic infection, liver fibrosis and malignant transformation, especially in recent years, with the in-depth study of the pathogenesis of HBV, the discovery of HBV in HBV The interaction with hepatocytes involves a complex trans regulation mechanism. The expression of hepatitis virus protein in liver cells affects the gene expression profiles of liver cells. This effect may be related to its pathogenesis. Therefore, it is necessary to search for the related genes in the liver cells to regulate the antigen of HBV antigens and further study it. The mechanism of action, effect and mutual influence are of great significance for identifying the pathogenesis of HBV and finding effective prevention and control methods. The cloning and expression and function of the selected unknown functional genes are the important source of innovative knowledge in the research field of viral hepatitis pathogenesis, and also the human genome project and the post basis. The important part of the group plan.
In this study, hepatocytes transfected with HBV -S1 protein gene fragment expressing vector and hepatocytes transfected only with empty carrier were used as material, and its mRNA application SSH technology was used to study the trans regulatory target gene of -S1 protein before HBV, and a unknown functional gene was screened, and the gene coding sequence was confirmed by dot blot technique. Hepatitis B virus -S1 protein trans regulatory protein 5 (PS1TP5), GenBankAccession:AY427953. discovery of a new gene, its new protein structure and biological function, the relationship with biological and clinical medicine, as well as the expression regulation mechanism is the most challenging work in the field of molecular biology. In this study, the following four aspects of the work have been done.
1. the HepG2 cell mRNA was extracted, and the EcoRI and Bgl II enzyme cutting sites were introduced at both ends of the PS1TP5 gene by RT-PCR method. The target gene fragment of 438bp was obtained and connected to pGEM-T vector. The sequence was correctly inserted into the prokaryotic expression vector pET-32a (+), and the BL21 host bacteria were transformed. The 1 mmol/LIPTG was induced at 30. Expression of recombinant fusion protein labeled with ammonia acid.
2. using ExPASy proteomics server to predict the physical and chemical properties and structural functions of PS1TP5, the relative molecular weight of.PS1TP5 is 15853.4, the theoretical pI is 9.41, and the extinction coefficients under different conditions are 17335 M~ (-1) cm~ (-1) or 16960 M~ (-1). The circumflex region, but has 4 hydrophobic regions, has no special two stage structure, and has 3 protein kinase phosphorylation sites, and does not have a transmembrane spiral structure, no signal peptide, and is located in the cytoplasm.
3. the PS1TP5 gene fragment was inserted into the yeast cell expression vector pGBKT7, the PS1TP5 gene fragment was inserted into the yeast cell expression vector pGBKT7, and the AH109 yeast strain was transformed and expressed. In conjunction with the Y187 yeast strain containing the leucocyte library plasmid, the yeast two hybrid screening was used to obtain the known functional proteins interacting with PS1TP5, including the white fine protein. Cell adhesion protein p150,95, interleukin 2 receptor gamma chain, PALM2-AKAP2 protein, eukaryotic translation initiation factor 4A, beta 2 microglobulin, sodium hydrogen exchange solute carrier family 9, non sialic acid glycoprotein receptor 1 (ASGR1), calcium reticulin, MHC type II type lymphocyte antigen, cytochrome C oxidase subtype 1, lymphocyte antigen 86 (LY86), lymphocyte solute The results suggest that PS1TP5 may be involved in hepatocyte proliferation, differentiation, information transmission and growth metabolism. In addition, a new gene is screened, named as -S1 protein trans activating protein 5 binding protein 1 (PS1TP5BP1), and GenBank accession as DQ471327..
4. the recombinant plasmid of pcDNA3.1/myc-His (-) A-PS1TP5 was constructed, and HepG2 cells were transfected with empty plasmids respectively. Both were parallel and controlled, and the transfected cells mRNA were extracted. The cDNA subtractive library of the differential expression of PS1TP5 protein trans activation related genes was successfully constructed, and 23 known trans up genes were screened and selected, including the signal conversion. Guide: 4 superfamily members of the family of transmembrane proteins (TM4SF1), 7 members of the solute carrier family 5 (SLC7A5), phosphoric inositase 2 (IMPA2), signal sequence receptor beta, epoxide 1, protein kinase BRPK, and protein factors: ribosomal protein S16, ribosomal protein LP0, ribosomal protein LP2, ribosomal protein S7, ribosome L31, and cluster protein transcription Mutant 1; transcription factor: eukaryotic extension factor 1; enzymes related to energy metabolism: NADH dehydrogenase 1, liver pyruvate dehydrogenase, cytochrome C oxidase subunit 8A, enolase 1, adenosine deaminase, etc. results suggest that PS1TP5 may be involved in human immune regulation, substance metabolism, signal transduction, especially with liver fibrosis and tumor development. There was a close relationship. In addition, a new gene was screened, named as the trans activator protein 1 (PS1TP5TP1) of the former -S1 protein trans activator protein 5 (PS1TP5TP1), and the GenBankaccession was DQ487761.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

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