2447nt-489nt乙型肝炎病毒剪接特异性蛋白与肝细胞蛋白相互作用研究

发布时间：2018-11-22 19:42

【摘要】： HBV基因组剪接变异体(spliced variants of hepatitis B virus genomes)是由HBV前基因组RNA (pregenomic RNA, pgRNA)经剪接并逆转录产生的亚基因组DNA。长度为2.2 kb的HBV剪接变异体又称为2447nt-489nt HBV剪接变异体占80%以上,可编码剪接特异性蛋白(Hepatitis B spliced protein, HBSP),并与病毒的持续性感染及致病性相关。本实验室先前利用酵母双杂交系统筛查肝细胞中与HBSP蛋白相互作用的细胞蛋白,分别获得纤维蛋白原γ链(fibrinogen gamma polypeptide, FGG)和微粒体环氧化物水解酶(microsomal epoxide hydrolase,mEH,编码基因为EPHX1)等几种候选蛋白。本研究在酵母双杂交系统初步筛查的基础上,进一步验证HBSP蛋白分别与FGG和mEH的相互作用,并探讨HBSP蛋白对患者凝血功能与肝脏生物转化功能的影响,从而探索其致病性。本研究第一部分旨在证实HBSP与FGG及mEH蛋白间的相互作用。为此,构建了pGEX-HBSP载体(用于GST-pull down)、pDsRed-HBSP载体(用于激光共聚焦)、pBIND-HBSP、pBIND-HBSP1-47和pBIND-HBSP64-111载体(用于哺乳动物细胞双杂交和免疫共沉淀);同时,采用RT-PCR技术从Huh7细胞扩增得到FGG和EPHX1全长基因并构建pCMVTNT-FGG和pCMVTNT-EPHX1载体(用于体外转录翻译)、pAcGFP-FGG和pAcGFP-EPHX1载体(用于激光共聚焦)。体外和体内的实验结果显示,HBSP蛋白可以和FGG或mEH直接相互作用,而且该相互作用由HBSP蛋白的N端47个氨基酸介导。本研究的第二部分旨在高效表达并获得高纯度HBSP融合蛋白,为此,将HBSP全长基因片段及其N端区域(编码N端47个氨基酸)和C端区域(编码N端64个氨基酸)分别克隆入原核表达载体pET43.1a(+)中,并在目的基因的C端引入可编码StrepII标签蛋白的基因片段,构建了pET-43-HBSP-StrepII、pET-43-HBSP1-47-StrepII、pET-43-HBSP48-111-StrepII和对照质粒pET-43-StrepII。之后应用IPTG诱导融合蛋白Nus-HBSP-StrepII、Nus-HBSP1-47-StrepII、Nus-HBSP48-111-StrepII和Nus-StrepII在Rosetta (DE3)中的表达。实验结果表明目的基因获得高效、可溶性表达,并通过Strep-Tactin系统亲和层析方法纯化得到高纯度的融合蛋白。本研究的第三部分探讨HBSP蛋白对FGG/mEH相关功能的影响。应用纯化后的融合蛋白进行纤维蛋白原凝集实验、血小板粘附实验和血小板聚集实验;结果表明HBSP蛋白可以通过与FGG结合抑制纤维蛋白原的凝集、干扰血小板对纤维蛋白原的粘附、激活和ADP引起的血小板聚集,从而抑制凝血功能,且HBSP蛋白的完整性对其功能至关重要;同时应用高效液相方法体外验证了完整的HBSP蛋白与mEH结合可大大增强mEH的酶活性,并促进苯并芘(Benzo(a)pyrene, BaP)的代谢,增加了最终致癌物Benzo(a)pyrene-7r, 8s-dihydrodiol-9s,10r-epoxide(±) (BPDE)的生成。结果提示HBSP蛋白可能抑制患者的凝血功能,与HBV感染者的出血倾向有关;HBSP蛋白可以促进多环芳香烃的代谢,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 纬 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 (卤) (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.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R373

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