【作者】 杨丹丽；

【导师】 周天鸿；

【作者基本信息】 暨南大学 ， 遗传学， 2010， 硕士

【摘要】 目的：人巨细胞病毒(HCMV)UL23基因,属病毒US22基因家族成员,编码病毒的皮层蛋白pUL23。目前pUL23蛋白的功能尚不清楚。我课题组通过酵母双杂交系统筛选了与人巨细胞病毒蛋白pUL23相互作用的宿主蛋白,其中IFP35蛋白是干扰素诱导蛋白。病毒感染过程会引起宿主免疫系统的反应,包括干扰素的反应,病毒蛋白pUL23与干扰素诱导蛋白IFP35的相互作用,可能与病毒激起宿主的免疫反应有关。为确定病毒蛋白pUL23是否与细胞干扰素途径相关,本课题通过GSTpull-down、免疫共沉淀及共定位等技术进一步确认pUL23与IFP35蛋白之间的相互作用,鉴定pUL23与IFP35相互作用蛋白结构域,以及pUL23与IFP35相互作用的生物学效应。研究成果将为揭示病毒蛋白pUL23的功能奠定基础。方法：1)pUL23与IFP35相互作用的确认：①酵母双杂交试验：将全长的UL23与IFP35分别构建于pGBKT7及pGADT7载体之上,利用酵母双杂交系统确定此两个蛋白的相互作用；②GST pull-down试验：构建pGEX-4T-1-IFP35载体并将其转化至大肠杆菌表达菌株Tuner(DE3)placI中表达纯化GST-IFP35蛋白,同时将pCDNA3.1(-)-HA-UL23载体转染Cos-7细胞,表达HA-UL23融合蛋白,将此两个蛋白共孵化后,用HA抗体进行Westenblotting检测；③免疫共沉淀试验(Co-IP)：构建pcDNA3.1(+)-IFP35-Flag载体,将其与pcDNA3.1(-)-HA-UL23共转染Cos-7细胞,分别用HA抗体和Flag抗体进行Westen blotting检测；④细胞共定位试验：构建pEGFP-N1-UL23和pDsRed2-C1-IFP35,将其共转染Hela细胞中,采用激光共聚焦显微镜观察蛋白在细胞内的定位情况。2)pUL23与IFP35相互作用结构域的鉴定：将27个不同长度的UL23短片断分别构建于pGBKT7及pGADT7载体之上,利用酵母双杂交系统来确定pUL23与蛋白相互作用的区域。紧接着将7个不同长度的IFP35短片断分别构建于pGBKT7及pGADT7载体之上,利用酵母双杂交系统来确定IFP35的作用区域。3)非变性聚丙烯酰胺凝胶电泳鉴定大分子复合物：pEGFP-N1-IFP35、pCMV-Nmi和pcDNA-HA-UL23分别转染到Hela细胞中,收集蛋白进行6%非变性聚丙烯酰胺凝胶电泳,Westen blotting检测结果。4) Westen blotting检测分子间相互关联：将pcDNA-HA-UL23、pcDNA3.1(+)、pcDNA-IFP35-Flag、pEGFP-N1按一定的质粒用量比例共转染到HEK293T细胞中,Westenblotting检测结果。结果：1)酵母双杂交系统,Co-IP, GST pull-down及共定位等四种方法共同证实了pUL23能与IFP35相互作用。2)通过共聚焦显微镜观察到病毒蛋白pUL23与IFP35共定位于细胞质,并处于细胞核周边。3)pUL23相互作用的重要区域在594-624bp之间,IFP35相互作用的重要区域在80-268aa之间,即两个串联的NID结构域。4)Nmi、IFP35、pUL23三者在Hela细胞中能形成一个大小约为300-400kDa的高分子聚合物。5)在HEK293细胞中,随着pUL23表达量的增加,IFP35表达量仅有微量的增加,IFP35的表达量影响pUL23的表达量,随着IFP35表达量的增加,pUL23表达量明显增加。结论：本课题运用酵母双杂交、Co-IP、GST pull-down及细胞共定位四种不同的实验技术确定HCMV pUL23与宿主蛋白IFP35相互作用。实验数据显示病毒蛋白pUL23与IFP35共定位于细胞质中,位于细胞核周边,pUL23与IFP35的NID结构域相互作用。在细胞内,pUL23和IFP35与另一个干扰素调控过程中重要蛋白Nmi形成大分子复合物,并且pUL23在细胞内的表达量与IFP35的表达量有关。以上实验结果提示了病毒蛋白pUL23与宿主细胞干扰素途径相关的分子之间存在密切的关系,为进一步研究病毒蛋白pUL23与宿主细胞免疫系统相关性提供了有力的证据。更多还原

【Abstract】 Object:Human cytomegalovirus (HCMV) UL23 gene encoding virus tegument protein pUL23 is a member of virus US22 gene family.Currently pUL23 function is unknown.My studied group screened interaction of host proteins with the human cytomegalovirus protein pUL23 by yeast two-hybrid experiment. One interactional protein is IFP35 which is a interferon inducible protein. Virus infection causes the host’s immune response, including the interferon response. It may be associated with the host immune response provoked by virus that interaction of viral protein pUL23 and interferon inducible protein IFP35. To determine whether the viral protein pUL23 relate to cellular interferon pathway, we use techniques of GST pull-down, immunoprecipitation and co-localization to further confirm protein interaction between pUL23 and IFP35, identificate interacting protein domain and the biological effects of interaction between pUL23 and IFP35. These results could provide the basis to study functions of pUL23.Methods:1) Determine the interaction between pUL23 and IFP35:①Yeast two-hybrid experiment: The full length of UL23 and IFP35 were respectively constructed in the pGBKT7 and pGADT7 vectors and then used yeast two-hybrid system to determine the interaction of these two proteins.②GST pull-down experiment:Constructed the pGEX-4T-1-IFP35 vector, transformed it into E. coli strain Tuner (DE3) placI to expressed and purified GST-IFP35 protein. The same time, transfected pCDNA3.1(-)-HA-UL23 vector into Cos-7 cells and expressed HA-UL23 fusion protein, after hatching these two proteins, Westen blotting with HA antibody were excuted.③Immunoprecipitation experiment (Co-IP):Construction of pcDNA3.1(+)-IFP35-Flag vector, the two expression vector pcDNA3.1(+)-IFP35-Flag and pcDNA3.1(-)-HA-UL23 co transfected into Cos-7 cell, Westen blotting with HA and Flag antibody were excuted.④Cells experiment of co-localization:Construction of pEGFP-Nl-UL23 and pDsRed2-C1-IFP35, these two expression vector were transfected into Hela cells, protein localization in cells by observated laser confocal microscope.2) Interaction domain of determining pUL23 and IFP35:27 different short pieces of UL23 were respectively constructed in the pGBKT7 and pGADT7 vector and then used yeast two-hybrid system to determine interactional region of pUL23, then the seven different IFP35 short pieces were respectively constructed in the pGBKT7 and pGADT7 vector, in order to confirme ineractional region of IFP35.3) Indentification of macromolecular complex by native polyacrylamide gel electrophoresis: pEGFP-N1-IFP35, pCMV-Nmi and pcDNA-HA-UL23 were transfected into Hela cells, supernatant fractions from cells were prepared and analyzed by Western blotting after running 6% native polyacrylamide gels.4) Westen blotting detect association among molecules:pcDNA-UL23-HA, pcDNA3.1(+), pcDNA-IFP35-Flag and pEGFP-N1 jointly transfected into HEK293T cells according to a certain percentage of plasmid dosage, Western blotting to test results.Result:1) Four methods of yeast two-hybrid system, Co-IP, GST pull-down and co-localization confirmed pUL23 can interact with IFP35.2) Co-localization of IFP35 and viral protein pUL23 is in the cytoplasm and surrounds nuclear observed by confocal microscopy.3) Important interactional regions of pUL23 are 594-624bp,while IFP35 are 80-268aa, that is NID domain.4) Nmi, IFP35 and pUL23 in Hela cells can form high polymer whoes size is about 300-400kDa.5) In HEK293 cells, with increase of the pUL23 expression, IFP35 expression was little increased.The expression of IFP35 influence the expression of pUL23. With increase of the IFP35 expression, pUL23 expression was obviously increased.Conclusion:My study determine the interaction between HCMV pUL23 and the host protein IFP35 by techniques of yeast two-hybrid system,GST pull-down, immunoprecipitation and co-localization. Experimental data show:Co-localization of IFP35 and viral protein pUL23 is in the cytoplasm and surrounds nuclear. pUL23 interact with the NID domain of IFP35. In the cells, pUL23, IFP35 and Nmi,which is another key interferon regulatory protein, form macromolecular. The expression of pUL23 in cells has relation with the expression of IFP35.The above mention results reveal the viral proteins pUL23 closely related to molecules involve interferon pathway in the host cell.It provided strong evidence to further study the relationship of viral proteins pUL23 and the immune system of host cells.更多还原