本文選題：乙型肝炎病毒 + 前-S1蛋白��； 參考：《山西醫(yī)科大學(xué)》2007年博士論文

【摘要】： 乙型肝炎病毒的持續(xù)感染至今仍然是一個(gè)全球性的健康問(wèn)題,不僅引起急慢性病毒性肝炎,而且與肝纖維化、肝細(xì)胞癌的發(fā)生發(fā)展密切相關(guān)。然而對(duì)于HBV感染目前尚無(wú)特效的治療方法,這主要是由于HBV感染引起病毒性肝炎的發(fā)病機(jī)制尚不清楚。目前認(rèn)為,HBV進(jìn)入肝細(xì)胞后,病毒基因組及其編碼的蛋白與肝細(xì)胞基因及蛋白之間的相互作用是決定HBV復(fù)制、表達(dá)、免疫逃逸、慢性感染、肝纖維化及致惡性轉(zhuǎn)化的關(guān)鍵因素。特別是近年來(lái)隨著對(duì)HBV致病機(jī)制研究的深入,發(fā)現(xiàn)在HBV與肝細(xì)胞相互作用的過(guò)程中涉及到復(fù)雜的反式調(diào)節(jié)機(jī)制,肝炎病毒蛋白在肝細(xì)胞中的表達(dá)對(duì)于肝細(xì)胞的基因表達(dá)譜產(chǎn)生影響,這一作用可能與其致病機(jī)制有關(guān)。因此,尋找肝細(xì)胞中與HBV各抗原成分反式調(diào)節(jié)的相關(guān)基因,并進(jìn)一步研究它的作用機(jī)制、作用效應(yīng)及相互影響,對(duì)明確HBV的致病機(jī)制,尋找有效防治方法具有重要意義。對(duì)于篩選得到的未知功能基因的克隆表達(dá)與功能研究是病毒性肝炎發(fā)病機(jī)制研究領(lǐng)域中創(chuàng)新知識(shí)的重要源泉,同時(shí)也是人類基因組計(jì)劃和后基因組計(jì)劃的重要內(nèi)容。 本研究利用轉(zhuǎn)染了HBV前-S1蛋白基因片段表達(dá)載體的肝細(xì)胞和僅轉(zhuǎn)染空載體的肝細(xì)胞為材料,提取其mRNA應(yīng)用SSH技術(shù)研究了HBV前-S1蛋白的反式調(diào)節(jié)靶基因,篩選得到一未知功能基因,經(jīng)斑點(diǎn)雜交技術(shù)驗(yàn)證后,確定其基因編碼序列,命名為乙型肝炎病毒前-S1蛋白反式調(diào)節(jié)蛋白5(PS1TP5),GenBankAccession:AY427953。發(fā)現(xiàn)一種新基因,將其編碼的新蛋白結(jié)構(gòu)與生物學(xué)功能,與生物學(xué)和臨床醫(yī)學(xué)之間的相互關(guān)系,以及表達(dá)調(diào)控機(jī)制闡明,是目前分子生物學(xué)研究領(lǐng)域中最具有挑戰(zhàn)性的工作。為此本研究做了以下四方面工作。 1.提取HepG2細(xì)胞mRNA,利用RT-PCR方法在PS1TP5基因的兩端分別引入EcoRI和BglⅡ酶切位點(diǎn),獲得了438bp的目的基因片段,連接至pGEM-T載體,測(cè)序正確后插入至原核表達(dá)載體pET-32a(+)中,轉(zhuǎn)化BL21宿主菌,1 mmol/LIPTG在30℃誘導(dǎo)2h、3h、4h后均獲得了帶有組氨酸標(biāo)簽的重組融合蛋白的表達(dá)。 2.利用ExPASy proteomics server對(duì)PS1TP5進(jìn)行蛋白理化性質(zhì)與結(jié)構(gòu)功能預(yù)測(cè)分析。PS1TP5相對(duì)分子量為15853.4,理論pI為9.41,不同條件下的消光系數(shù)為17335 M~(-1)cm~(-1)或16960 M~(-1)cm~(-1)(280nm),在體外哺乳動(dòng)物網(wǎng)狀細(xì)胞中的半壽期為30hr,并且無(wú)卷曲螺旋區(qū)域,但具有4個(gè)疏水區(qū);無(wú)特殊二級(jí)結(jié)構(gòu),有3個(gè)蛋白激酶磷酸化位點(diǎn),不具有跨膜螺旋結(jié)構(gòu),無(wú)信號(hào)肽,定位于細(xì)胞漿中。 3.將引入EcoRI和BglⅡ酶切位點(diǎn)的PS1TP5基因片段插入至酵母細(xì)胞表達(dá)載體pGBKT7中,轉(zhuǎn)化AH109酵母菌株并獲得表達(dá),與含有白細(xì)胞文庫(kù)質(zhì)粒的Y187酵母菌株配合,進(jìn)行酵母雙雜交篩選獲得了與PS1TP5具有相互作用的已知功能蛋白23種,包括白細(xì)胞黏附蛋白p150,95、白介素2受體γ鏈、PALM2-AKAP2蛋白、真核翻譯起始因子4A、β2微球蛋白、鈉-氫交換溶質(zhì)載體家族9、無(wú)唾液酸糖蛋白受體1(ASGR1)、鈣網(wǎng)織蛋白、MHC類Ⅱ型淋巴細(xì)胞抗原、細(xì)胞色素C氧化酶亞型1、淋巴細(xì)胞抗原86(LY86)、淋巴細(xì)胞溶質(zhì)蛋白1等。結(jié)果提示PS1TP5可能參與肝細(xì)胞增殖分化、信息傳遞以及生長(zhǎng)代謝。此外還篩選得到一個(gè)新基因,命名為乙型肝炎病毒前-S1蛋白反式激活蛋白5結(jié)合蛋白1(PS1TP5BP1),GenBank accession為DQ471327。 4.構(gòu)建pcDNA3.1/myc-His(-)A-PS1TP5真核表達(dá)重組質(zhì)粒,與空質(zhì)粒分別轉(zhuǎn)染HepG2細(xì)胞,兩者互為平行對(duì)照,提取轉(zhuǎn)染后的細(xì)胞mRNA,成功地構(gòu)建了PS1TP5蛋白反式激活相關(guān)基因差異表達(dá)的cDNA消減文庫(kù),篩選獲得了23條已知的反式上調(diào)基因,包括信號(hào)轉(zhuǎn)導(dǎo)分子:跨膜蛋白家族4超家族成員1(TM4SF1)、溶質(zhì)載體家族7成員5(SLC7A5)、磷酸肌醇酶2(IMPA2)、信號(hào)序列受體-β、環(huán)氧化物酶1、蛋白激酶BRPK等;蛋白因子:核糖體蛋白S16、核糖體蛋白LP0、核糖體蛋白LP2、核糖體蛋白S7、核糖體蛋白L31、叢生蛋白轉(zhuǎn)錄突變體1等;轉(zhuǎn)錄因子:真核延伸因子1;能量代謝有關(guān)的酶:NADH脫氫酶1、肝丙酮酸脫氫酶、細(xì)胞色素C氧化酶亞單位8A、烯醇酶1、腺苷脫氨酶等。結(jié)果提示PS1TP5可能參與人類免疫調(diào)節(jié)、物質(zhì)代謝、信號(hào)轉(zhuǎn)導(dǎo)。特別是與肝纖維化形成、腫瘤發(fā)生發(fā)展有密切關(guān)系。此外還篩選得到一個(gè)新基因,命名為乙型肝炎病毒前-S1蛋白反式激活蛋白5反式激活蛋白1(PS1TP5TP1),GenBankaccession為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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 劉妍 ,董菁 ,皇甫競(jìng)坤 ,成軍 ,王琳 ,王剛 ,李莉;乙型肝炎病毒X基因異質(zhì)性及對(duì)其反式激活功能的影響[J];解放軍醫(yī)學(xué)雜志;2002年02期

2 紀(jì)冬,成軍,王建軍,董菁,郭江,劉妍,楊倩,黨曉燕,王春花;應(yīng)用抑制性消減雜交技術(shù)克隆乙型肝炎病毒前-S1蛋白反式激活的相關(guān)基因[J];胃腸病學(xué)和肝病學(xué)雜志;2004年01期

3 成軍;;新基因結(jié)構(gòu)與功能研究的策略[J];世界華人消化雜志;2003年04期

4 成軍;劉妍;陸蔭英;李克;王琳;;乙型肝炎病毒對(duì)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)的影響[J];世界華人消化雜志;2003年04期

5 成軍;劉妍;王琳;鐘彥偉;王剛;;乙型和丙型肝炎病毒蛋白對(duì)于細(xì)胞周期素A的調(diào)節(jié)研究[J];世界華人消化雜志;2003年08期

6 王春花,成軍,郎振為,李蘊(yùn)茹,閆杰,張黎穎;乙型肝炎病毒前-前-S蛋白在大腸桿菌中的表達(dá)和純化[J];世界華人消化雜志;2005年15期

7 劉妍,成軍,王剛,李克,段惠娟,王琳,李莉,張玲霞,陳菊梅;HCV核心蛋白與截短型HBV表面抗原中蛋白的協(xié)同反式激活功能[J];中華肝臟病雜志;2002年05期

8 孫秀菊;孫開來(lái);鄭志紅;富偉能;郝冬梅;徐惠綿;李曉明;;腸型胃癌不同發(fā)展階段基因表達(dá)譜分析(英文)[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2006年02期

9 李凌,馬文麗,鄭文嶺,徐鈐;PROGRESS IN DNA CHIP TECHNOLOGY[J];Chinese Medical Sciences Journal;2001年01期

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