【摘要】： HBV基因組剪接變異體(spliced variants of hepatitis B virus genomes)是由HBV前基因組RNA (pregenomic RNA, pgRNA)經(jīng)剪接并逆轉(zhuǎn)錄產(chǎn)生的亞基因組DNA。長(zhǎng)度為2.2 kb的HBV剪接變異體又稱為2447nt-489nt HBV剪接變異體占80%以上,可編碼剪接特異性蛋白(Hepatitis B spliced protein, HBSP),并與病毒的持續(xù)性感染及致病性相關(guān)。本實(shí)驗(yàn)室先前利用酵母雙雜交系統(tǒng)篩查肝細(xì)胞中與HBSP蛋白相互作用的細(xì)胞蛋白,分別獲得纖維蛋白原γ鏈(fibrinogen gamma polypeptide, FGG)和微粒體環(huán)氧化物水解酶(microsomal epoxide hydrolase,mEH,編碼基因?yàn)镋PHX1)等幾種候選蛋白。本研究在酵母雙雜交系統(tǒng)初步篩查的基礎(chǔ)上,進(jìn)一步驗(yàn)證HBSP蛋白分別與FGG和mEH的相互作用,并探討HBSP蛋白對(duì)患者凝血功能與肝臟生物轉(zhuǎn)化功能的影響,從而探索其致病性。 本研究第一部分旨在證實(shí)HBSP與FGG及mEH蛋白間的相互作用。為此,構(gòu)建了pGEX-HBSP載體(用于GST-pull down)、pDsRed-HBSP載體(用于激光共聚焦)、pBIND-HBSP、pBIND-HBSP1-47和pBIND-HBSP64-111載體(用于哺乳動(dòng)物細(xì)胞雙雜交和免疫共沉淀);同時(shí),采用RT-PCR技術(shù)從Huh7細(xì)胞擴(kuò)增得到FGG和EPHX1全長(zhǎng)基因并構(gòu)建pCMVTNT-FGG和pCMVTNT-EPHX1載體(用于體外轉(zhuǎn)錄翻譯)、pAcGFP-FGG和pAcGFP-EPHX1載體(用于激光共聚焦)。體外和體內(nèi)的實(shí)驗(yàn)結(jié)果顯示,HBSP蛋白可以和FGG或mEH直接相互作用,而且該相互作用由HBSP蛋白的N端47個(gè)氨基酸介導(dǎo)。 本研究的第二部分旨在高效表達(dá)并獲得高純度HBSP融合蛋白,為此,將HBSP全長(zhǎng)基因片段及其N端區(qū)域(編碼N端47個(gè)氨基酸)和C端區(qū)域(編碼N端64個(gè)氨基酸)分別克隆入原核表達(dá)載體pET43.1a(+)中,并在目的基因的C端引入可編碼StrepII標(biāo)簽蛋白的基因片段,構(gòu)建了pET-43-HBSP-StrepII、pET-43-HBSP1-47-StrepII、pET-43-HBSP48-111-StrepII和對(duì)照質(zhì)粒pET-43-StrepII。之后應(yīng)用IPTG誘導(dǎo)融合蛋白Nus-HBSP-StrepII、Nus-HBSP1-47-StrepII、Nus-HBSP48-111-StrepII和Nus-StrepII在Rosetta (DE3)中的表達(dá)。實(shí)驗(yàn)結(jié)果表明目的基因獲得高效、可溶性表達(dá),并通過(guò)Strep-Tactin系統(tǒng)親和層析方法純化得到高純度的融合蛋白。 本研究的第三部分探討HBSP蛋白對(duì)FGG/mEH相關(guān)功能的影響。應(yīng)用純化后的融合蛋白進(jìn)行纖維蛋白原凝集實(shí)驗(yàn)、血小板粘附實(shí)驗(yàn)和血小板聚集實(shí)驗(yàn);結(jié)果表明HBSP蛋白可以通過(guò)與FGG結(jié)合抑制纖維蛋白原的凝集、干擾血小板對(duì)纖維蛋白原的粘附、激活和ADP引起的血小板聚集,從而抑制凝血功能,且HBSP蛋白的完整性對(duì)其功能至關(guān)重要;同時(shí)應(yīng)用高效液相方法體外驗(yàn)證了完整的HBSP蛋白與mEH結(jié)合可大大增強(qiáng)mEH的酶活性,并促進(jìn)苯并芘(Benzo(a)pyrene, BaP)的代謝,增加了最終致癌物Benzo(a)pyrene-7r, 8s-dihydrodiol-9s,10r-epoxide(±) (BPDE)的生成。結(jié)果提示HBSP蛋白可能抑制患者的凝血功能,與HBV感染者的出血傾向有關(guān);HBSP蛋白可以促進(jìn)多環(huán)芳香烴的代謝,加速了致癌物的致癌作用。
[Abstract]:HBV genomic splicing variant (spliced variants of hepatitis B virus genomes) is a subgenomic DNA. produced by splicing and reverse transcription of pre-genomic RNA (pregenomic RNA, pgRNA) of HBV. More than 80% of HBV splicing variants with a length of 2.2 kb, also known as 2447nt-489nt HBV splicing variants, can encode splicing specific protein (Hepatitis B spliced protein, HBSP), and are associated with persistent infection and pathogenicity of the virus. Previously, yeast two-hybrid system was used to screen cellular proteins interacting with HBSP protein in hepatocytes. Fibrinogen 緯 chain (fibrinogen gamma polypeptide, FGG) and microsomal epoxide hydrolase (microsomal epoxide hydrolase,mEH, were obtained, respectively. Coding base because of EPHX1) and other candidate proteins. Based on the screening of yeast two-hybrid system, the interaction of HBSP protein with FGG and mEH was further verified, and the effect of HBSP protein on coagulation function and liver biotransformation function of patients was discussed, so as to explore its pathogenicity. The first part of this study was to confirm the interaction of HBSP with FGG and mEH proteins. Therefore, pGEX-HBSP vectors (for laser confocal laser), pBIND-HBSP,pBIND-HBSP1-47 and pBIND-HBSP64-111 vectors (for mammalian cell two-hybrid and immunoprecipitation) were constructed. At the same time, the full-length FGG and EPHX1 genes were amplified by RT-PCR from Huh7 cells and constructed pCMVTNT-FGG and pCMVTNT-EPHX1 vectors (for in vitro transcriptional translation), pAcGFP-FGG and pAcGFP-EPHX1 vectors (for laser confocal). The results in vitro and in vivo showed that HBSP protein could interact directly with FGG or mEH, and the interaction was mediated by 47 amino acids at the N-terminal of HBSP protein. The second part of this study aims to express and obtain high purity HBSP fusion protein. The full-length gene fragment of HBSP and its N-terminal region (encoding 47 amino acids at N-terminal) and C-terminal region (encoding 64 amino acids at N-terminal) were cloned into prokaryotic expression vector pET43.1a (), respectively. A gene fragment encoding StrepII tag protein was introduced into the C terminal of the target gene, and pET-43-HBSP-StrepII,pET-43-HBSP1-47-StrepII,pET-43-HBSP48-111-StrepII and control plasmid pET-43-StrepII. were constructed. Then IPTG was used to induce the expression of Nus-HBSP-StrepII,Nus-HBSP1-47-StrepII,Nus-HBSP48-111-StrepII and Nus-StrepII in Rosetta (DE3). The results showed that the target gene was expressed efficiently and soluble, and the fusion protein was purified by Strep-Tactin affinity chromatography. The third part of this study was to investigate the effect of HBSP protein on FGG/mEH related functions. The purified fusion protein was used for fibrinogen agglutination test, platelet adhesion test and platelet aggregation test. The results showed that HBSP protein could inhibit coagulation function by inhibiting fibrinogen agglutination, interfering platelet adhesion to fibrinogen, activation and platelet aggregation induced by ADP by binding with FGG. The integrity of HBSP protein is very important to its function. At the same time, high performance liquid phase method was used to verify that the binding of complete HBSP protein to mEH could greatly enhance the enzyme activity of mEH, promote the metabolism of benzopyrene (Benzo (a) pyrene, BaP), and increase the final carcinogen Benzo (a) pyrene-7r, 8s-dihydrodiol-9s. The generation of 10r-epoxide (鹵) (BPDE). The results suggest that HBSP protein may inhibit the coagulation function of patients with HBV, and HBSP protein can promote the metabolism of polycyclic aromatic hydrocarbons and accelerate the carcinogenic effect of carcinogens.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R373

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張偉;黃旭東;韓小波;任珊珊;王小柯;李艷艷;唐勝建;;FOXL2酵母雙雜交誘餌載體構(gòu)建及檢測(cè)[J];生物技術(shù);2011年03期

2 張泉;王珍;翟國(guó)勤;薛整風(fēng);徐向明;;酵母雙雜交系統(tǒng)對(duì)CDV H結(jié)合蛋白的初步篩選[J];江蘇農(nóng)業(yè)學(xué)報(bào);2011年04期

3 李向陽(yáng);張嘉保;;流產(chǎn)型布魯氏菌Omp22基因酵母雙雜交誘餌載體的構(gòu)建及鑒定[J];中國(guó)畜牧獸醫(yī);2011年06期

4 余靜;羅韜;羅美中;;利用酵母雙雜交篩選豌豆葉綠體鎂離子螯合酶D亞基相互作用蛋白[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);2011年09期

5 張洪興;金毅;茍萍;王星;藍(lán)柯;;卡波氏肉瘤相關(guān)皰疹病毒ORF50結(jié)合蛋白的篩選[J];上海師范大學(xué)學(xué)報(bào)(自然科學(xué)版);2011年03期

6 劉菊華;徐碧玉;張靜;張建斌;賈彩紅;金志強(qiáng);;MADS-box基因酵母雙雜交系統(tǒng)的構(gòu)建及鑒定[J];熱帶作物學(xué)報(bào);2011年07期

7 羅瑋;周天鴻;閆道廣;;ORP8與SPAG5相互作用并影響細(xì)胞周期[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2011年14期

8 許奕;金志強(qiáng);劉菊華;賈彩虹;張建斌;徐碧玉;;asr基因酵母雙雜交系統(tǒng)的構(gòu)建及篩選鑒定[J];生物技術(shù)通報(bào);2011年09期

9 葉曉霞;石彥;霍克克;陳東;吳民華;;羧基末端結(jié)合蛋白CtBP2與泛素結(jié)合酶UBE2Ⅰ的相互作用[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2011年17期

10 楊潔;趙亞力;伍志強(qiáng);韓為東;;Macro Domain家族成員LRP16是多種核受體的相互作用因子[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);2011年08期

相關(guān)會(huì)議論文 前10條

1 符向輝;何淑雅;董曉;李斌元;王三虎;秦凌雪;陳小衛(wèi);馬云;;智力低下相關(guān)蛋白FXR1P與CMAS相互作用區(qū)域的鑒定[A];傳承與發(fā)展，，創(chuàng)湖南省生理科學(xué)事業(yè)的新高——湖南省生理科學(xué)會(huì)2011年度學(xué)術(shù)年會(huì)論文摘要匯編[C];2011年

2 李玉花;聶玉哲;張e

本文編號(hào)：2350345