ATPase 抑制因子1是与HCMV pUL23蛋白相作用的宿主蛋白分子-酵母双杂交技术筛选与鉴定

目的： 利用酵母双杂交技术筛选与人巨细胞病毒相互作用的宿主蛋白分子,为探讨人巨细胞病毒pUL23蛋白在HCMV生活周期中的作用机制提供依据。方法: 利用GAL4酵母双杂交系统筛选人胚肾cDNA文库,以获得与人巨细胞病毒pUL23蛋白相互作用的宿主蛋白分子,再通过回交试验和体外GST-pulldown试验验证两者之间的相互作用。结果: 酵母双杂交筛选得到宿主蛋白分子ATPase inhibitory factor 1（ATIF1）,回交试验和体外GST-pulldown试验再次确认ATIF1能够与人巨细胞病毒pUL23蛋白相互作用。结论: pUL23确实能够与ATIF1相互作用,它们之间的相互作用可能为研究pUL23在病毒生活周期发挥的功能提供依据。     

稳定表达人巨细胞病毒蛋白pUL23细胞系建立及其功能研究

目的:建立稳定表达人巨细胞病毒UL23基因的HELF细胞系,研究病毒蛋白在宿主细胞内行为,为进一步研究人巨细胞病毒蛋白pUL23的功能提供依据。方法:通过PCR技术从人巨细胞病毒基因组中扩增出UL23基因,通过分子克隆技术构建重组逆转录病毒表达载体pLEGFP-N1-FLAG-UL23。将该载体导入Am-phoPackTM-293细胞,收获重组逆转录病毒,然后感染HELF细胞,HELF经持续G418抗性筛选后获得稳定表达UL23基因的细胞系。采用激光共聚焦显微镜观察病毒蛋白在细胞内的定位。结果:RT-PCR、Western blotting结果证实病毒基因UL23能够整合到宿主细胞基因组中,并能在宿主细胞中稳定表达病毒蛋白。共聚焦显微镜观察到病毒蛋白pUL23定位于细胞质,处于细胞核周边。结论:利用逆转录病毒载体介导的基因转移技术,成功构建了稳定表达UL23基因的转基因细胞系。该病毒蛋白在宿主细胞质中的定位,提示病毒蛋白发挥功能的空间位于细胞核周边,有利地推进了人巨细胞病毒蛋白pUL23功能研究的进程。     

MIF活性区域相互作用蛋白的筛选

目的: 筛选与巨噬细胞移动抑制因子(MIF)催化巯基蛋白质氧化还原酶(TPOR)活性域相互结合的蛋白。方法: 采用酵母双杂交系统进行筛选。首先构建pBTM116-MIF诱饵质粒,转化酵母菌株L40;将表达MIF催化 TPOR活性域的L40酵母菌株制备成感受态菌,在人骨肉瘤cDNA文库中筛选。通过组氨酸(HIS3报告基因活性和β半乳糖苷酶实验,初步确定与催化TPOR活性域片段相互作用的蛋白,最终通过免疫共沉淀和免疫荧光技术来确认。结果: 分别构建含野生型MIF的pBTM116-MIF和野生型硫氧还蛋白样蛋白2(TXNL2)的pACT2-TXNL2质粒,将其共转染L40酵母菌。HIS3报告基因活性检测和β半乳糖苷酶阳性实验结果提示TXNL2可能是与MIF催化 TPOR活性片段相互作用的蛋白。将重组质粒pcDNA3.1-Myc-TXNL2转染MCF7细胞,免疫共沉淀实验结果表明MIF抗体能够共沉淀Myc-TXNL2蛋白;免疫荧光结果显示Myc-TXNL2与MIF在细胞质中位置分布相同。结论: TXNL2可能是与MIF催化TPOR活性片段相互作用的蛋白。本研究为MIF氧化还原机制的研究提供了新的实验依据。     

干扰素诱导蛋白Nmi与人巨细胞病毒皮层蛋白UL23相互作用区域的研究

目的:探究干扰素诱导蛋白N-Myc相互作用因子(Nmi)与人巨细胞病毒(HCMV)皮层蛋白UL23相互作用的关键区域。方法:根据前期实验结果,分别将10个不同截短突变型的Nmi构建到原核表达载体p GEX-4T-1上,转化到大肠杆菌Rosetta(DE3)感受态细胞中,表达并纯化出带有GST标签的融合蛋白,利用GST-pulldown的方法探究Nmi与UL23相互作用的区域。根据GST-Pulldown实验结果,用同源重组的方法在真核表达载体pc DNA4-Myc上分别构建3个缺失突变型Nmi。将实验组和对照组的Nmi分别与含有Flag标签的UL23质粒共转染至He La细胞中,通过免疫共沉淀法进一步研究Nmi与UL23的相互作用区域。结果:(1)10个截短突变型Nmi与GST基因融合的原核表达载体构建成功;(2)3个缺失突变型Nmi与Myc基因融合的真核表达载体构建成功;(3)GST-pulldown实验证明Nmi与UL23相互作用位点位于Nmi上的第192~202位氨基酸区域;(4)免疫共沉淀法确认Nmi的第192~202位氨基酸区域是与UL23相互作用的区域,与GST-pulldown实验结果一致。结论:Nmi与UL23相互作用的区域位于Nmi上第192~202位氨基酸区域。这为阐明UL23帮助HCMV在宿主体内潜伏的分子机理提供了基础。     

与接头蛋白Bam32相互作用的蛋白分子的鉴定

目的:研究接头蛋白(adaptorprotein)Bam32在B细胞抗原受体(Bcellantigenreceptor,BCR)信号转导级联反应中的作用。方法:以Bam32全序列为诱饵,应用酵母双杂交技术筛选能与Bam32相互作用的蛋白分子,并用293T细胞共转染和免疫共沉淀法加以证实。结果:应用酵母双杂交技术筛选出能与Bam32相互作用的蛋白分子,其中1个出现强阳性反应的克隆编码酪氨酸激酶Lyn。用293T细胞共转染和特异性免疫共沉淀法证实,Bam32可在哺乳动物细胞中与Lyn共沉淀。应用抗磷酸化酪氨酸抗体检测显示,Bam32与Lyn相互作用可导致Bam32磷酸化。结论:Bam32可同Lyn相互作用,导致Bam32磷酸化,这在激活下游产物的级联反应中可能起重要作用。     

原癌基因LMO3相互作用蛋白的筛选及鉴定

目的 通过筛选LMO3的相互作用蛋白,进一步了解LMO3的作用及可能机制.方法:酵母双杂交方法 筛选LMO3相互作用蛋白,并通过酵母结合试验、免疫共沉淀及荧光共定位等进行验证.结果:在初步获得相互作用蛋白CIB的基础上,在酵母中证实了LMO3与CIB的相互作用,并通过酵母结合试验确定了CIB与LMO3的相互作用位点,发...     

酵母双杂交系统筛选GATA-1相互作用蛋白质及功能验证

目的 利用酵母双杂交技术从人脑cDNA文库中筛选与人GATA-1相互作用的蛋白质.方法 从人K562细胞中扩增出全长GATA1基因,设计引物将其3段截断体亚克隆入酵母表达载体pDBLeu中,转化至AH109感受态酵母中,利用酵母双杂交技术筛选人脑cDNA文库中与其相互作用的蛋白质,阳性克隆通过回转及免疫共沉淀试验进行验证,利用3xGATA荧光素酶报告基因对相互作用蛋白质进行功能验证.结果 成功构建出酵母诱饵蛋白表达质粒pDBLeu-GATA1(1),pDBLeu-GATA1(2),pDBLeu-GATA1(3),筛到34个阳性克隆,用生物信息学分析及回转验证得到5个与GATA-1相互作用的候选蛋白,通过免疫共沉淀试验进一步验证,获得3个蛋白质能与GATA-1相互作用,分别是ECSIT,EFEMP1和GPS2.荧光素酶试验表明这3个蛋白质均能对GATA1的转录活性产生影响,证实它们之间的相互作用具有影响GATA1转录的功能.结论 应用酵母双杂交技术及免疫共沉淀试验,从人脑cDNA文库中成功获得3个与GATA-1相互作用并对其转录活性具有调节作用的蛋白质,为研究GATA1蛋白质的功能提供了新的线索.     

应用酵母双杂交系统筛选人胃黏膜上皮组织SHIP2的相互作用蛋白

目的通过酵母双杂交系统筛选人胃黏膜上皮组织标准均一化cDNA文库,寻找与含Src同源蛋白2肌醇-5-磷酸酶2(SHIP2)相互作用的蛋白。方法利用酵母双杂交系统,以SHIP2的P1(SH2+5-Ptase)和P2(PRD+SAM)段作为诱饵蛋白,筛选出人胃黏膜上皮组织均一化cDNA文库中与SHIP2相互作用的蛋白,并通过免疫共沉淀法进行验证。结果挑选出39个阳性克隆,经测序比对分析,回复性杂交,免疫共沉淀试验验证,最终确定一个与SHIP2相互作用的蛋白抗增殖蛋白1(prohibitin1/PHB)。结论酵母双杂交系统筛选人胃黏膜上皮组织SHIP2的相互作用蛋白PHB。     

红系分化相关基因相互作用蛋白质的分离与鉴定

目的制备红系分化相关基因(erythroid differentiation-associated gene,EDAG)蛋白的单克隆抗体,利用免疫沉淀联合质谱技术对EDAG相互作用蛋白质进行分离与鉴定。方法构建EDAG原核表达载体,通过诱导、表达、纯化获得EDAG融合蛋白,杂交瘤技术建立分泌EDAG单克隆抗体的杂交瘤细胞株,利用Western印迹筛选阳性杂交瘤细胞,免疫小鼠得腹水,最后用免疫共沉淀与质谱联合鉴定EDAG相互作用蛋白。结果纯化获得EDAG重组蛋白,筛选出4株分泌EDAG单克隆抗体的杂交瘤细胞株,并制备了腹水。该抗体均可用于内源EDAG蛋白的检测,其中两种抗体可用于免疫共沉淀实验,运用质谱技术筛选获得EDAG候选相互作用蛋白。结论利用EDAG单克隆抗体,筛选到EDAG候选相互作用蛋白质13种,涉及细胞增殖及转录等过程,为EDAG的功能研究及其分子机制的阐明提供了新的线索。     

Interactions between human cytomegalovirus helicase-primase proteins

The human cytomegalovirus (HCMV) UL70, UL102, and UL105 genes are predicted to encode essential proteins that assemble the replicative helicase-primase complex based on sequence and genome position similarities to putative herpes simplex virus type 1 (HSV-1) counterparts. Consistent with this prediction, they are required for transient complementation of DNA synthesis. However, little is known about their physical interactions and biochemical activities, primarily because of their restricted expression in HCMV-infected cells. To look for assembly of the predicted complexes, we prepared rabbit polyclonal antisera and used Semliki Forest Virus (SFV) vectors to express untagged and glutathione-S-transferase (GST)-tagged UL70, UL102 and UL105 proteins. The UL70 and UL105 proteins co-purified with the GST-tagged UL102 protein from triply-infected baby hamster kidney cells (BHK-21), and pUL70, but not pUL105, co-purified with pGST-UL102 from dually infected BHK-21 cells. In immunoprecipitation experiments with untagged SFV-expressed proteins, pUL70 or pUL105 coprecipitated with pUL102, pUL102 or pUL70 co-precipitated with pUL105; and pUL102 or pUL105 coprecipitated with pUL70. Comparison of the GST-pull down and immunoprecipitation experiments suggested that the amino-terminal GST-tag interfered with certain pairwise interactions. These results support the prediction that the HCMV helicase-primase proteins assemble a three-protein heteromeric complex, and show that each protein contacts both partners.     

The sequence and antiapoptotic functional domains of the human cytomegalovirus UL37 exon 1 immediate early protein are conserved in multiple primary strains

The human cytomegalovirus UL37 exon 1 gene encodes the immediate early protein pUL37x1 that has antiapoptotic and regulatory activities. Deletion mutagenesis analysis of the open reading frame of UL37x1 identified two domains that are necessary and sufficient for its antiapoptotic activity. These domains are confined within the segments between amino acids 5 to 34, and 118 to 147, respectively. The first domain provides the targeting of the protein to mitochondria. Direct PCR sequencing of UL37 exon 1 amplified from 26 primary strains of human cytomegalovirus demonstrated that the promoter, polyadenylation signal, and the two segments of pUL37x1 required for its antiapoptotic function were invariant in all sequenced strains and identical to those in AD169 pUL37x1. In total, UL37 exon 1 varies between 0.0 and 1.6% at the nucleotide level from strain AD169. Only 11 amino acids were found to vary in one or more viral strains, and these variations occurred only in the domains of pUL37x1 dispensable for its antiapoptotic function. We infer from this remarkable conservation of pUL37x1 in primary strains that this protein and, probably, its antiapoptotic function are required for productive replication of human cytomegalovirus in humans.     

Molecular dissection of the human B cell response against cytomegalovirus infection by lambda display

Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, is the main cause of congenital abnormalities and mental retardation in newborns and is also responsible for severe life-threatening complications in immunocompromised individuals, including AIDS patients and transplant recipients. The disorders generated by cytomegalovirus are closely associated with the competence of the host immune system and both humoral and cell-mediated mechanisms are involved in the response to viral infection. To identify viral proteins recognized by host antibody responses, a cytomegalovirus genome library was created and displayed on lambda bacteriophage. The challenge of such a library with sera from individuals with congenital or acquired infection allowed the identification of a wide panel of recombinant bacteriophages carrying cytomegalovirus B cell epitopes. Epitope-containing fragments within the families of tegument proteins (pUL25, pUL32), structural proteins (pUL48, pUL56) and glycoproteins (pUL55) were identified. Moreover, library screening permitted isolation of phage clones carrying an antigenic region of an uncharacterized HCMV protein encoded by the UL71 open reading frame (ORF), highlighting the potential of lambda display technology in antigen and epitope discovery.     

Characterization of the guinea pig cytomegalovirus genome locus that encodes homologs of human cytomegalovirus major immediate-early genes, UL128, and UL130

We reported previously that the guinea pig cytomegalovirus (CMV) stock purchased from the American Type Culture Collection contained two types of strains, one containing and the other lacking a 1.6 kb locus, and that the 1.6 kb locus was required for efficient viral growth in animals but not in cell culture. In this study, we characterized the genetic contents of the locus, and found that i) the 1.6 kb locus encodes homologs of human CMV UL128 and UL130, GP129 and GP131, respectively, ii) these genes are expressed with late gene kinetics, iii) GP131 protein (pGP131) localized to cell surface only in the presence of glycoproteins H and L, and iv) pGP131 is a virion component. Therefore, it is plausible that pGP131 forms a complex with glycoproteins H and L and becomes a virion component as does UL130 protein (pUL130). Since pUL130 is one of the glycoproteins essential for infection of endothelial and epithelial cells in human and primates, functional and immunological analyses of this GPCMV homolog of pUL130 may help to illuminate the in vivo role of pUL130.     

A rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus

We have developed a murine monoclonal antibody reactive with a major immediate early 72,000 dalton protein of human cytomegalovirus and utilized this reagent in a rapid virus titration and microneutralization assay. Because of the early expression of this virus encoded protein, both assays could be accomplished within 16 h following virus inoculation. In addition, both assays resulted in considerable savings of reagents because the assays were carried out in 96-well microtiter plates. These assays should prove useful in the preparation and study of neutralizing antibodies directed against human cytomegalovirus.     

The product of human cytomegalovirus UL73 is a new polymorphic structural glycoprotein (gpUL73)

OBJECTIVES: This work focuses on human cytomegalovirus (HCMV) UL73, which encodes for a putative transmembrane glycoprotein that is highly conserved among herpesviruses. STUDY DESIGN: pUL73 expression was analyzed both in transiently transfected and in HCMV-infected cells using a pUL73-specific antiserum by immunoblot and immunofluorescence. Sequencing analysis from several clinical isolates and laboratory-adapted strains was also performed. RESULTS: pUL73 expressed in transiently transfected cells consists in a polypeptide of the expected size (15-18 kd) with cytoplasmic localization. In infected cells, pUL73 is expressed with true-late kinetics and localizes both in perinuclear granular formations and on the cell surface. A broad band (39-53 kd), sensitive to O-glycosidase digestion was detected in purified virus. In addition, sequence analysis showed that the N-terminal portion of pUL73 from clinical isolates is highly polymorphic. CONCLUSIONS: UL73 encodes for a new structural glycoprotein (gpUL73) expressed on the cell surface of infected cells and highly polymorphic among clinical isolates.     

pUL25 immunolocalization in human cytomegalovirus-infected and gene-transfected cells

Summary.  By means of confocal and electron microscope immunocytochemistry we have studied the localization of a recently described structural protein (pUL25) of human cytomegalovirus, in both infected cells and in cells transiently transfected with UL25. pUL25 localization in infected cells was observed in typical cytoplasmic structures characterized by a very electrondense texture previously reported to accumulate other tegument proteins. At the virion level pUL25 seems to localize at the interface between the tegument and the capsid of both intracytoplasmic and extracellular virions. In UL-25-transfected cells, pUL25 has been found in characteristic para-crystalline cytoplasmic aggregates, suggesting its intrinsic ability to aggregate in a regular subunit pattern. Received August 6, 1999/Accepted November 3, 1999