人巨细胞病毒gH蛋白表达及其单克隆抗体的制备

人巨细胞病毒(HCMV)是全球范围内普遍感染的一种疱疹病毒,也是引起胎儿先天性畸形和器官移植受者死亡最常见的病原体。有研究表明,gH蛋白是HCMV主要的中和性抗原,特异性抗gH抗体具有中和HCMV及阻断病毒在细胞间传播的潜力。本研究以含人巨细胞病毒临床株Toledo全基因组的细菌人工染色体(BAC)为模板,扩增去除信号肽和跨膜区的gH基因片段,并将其插入表达载体构建pET32a'-gH重组表达质粒,将序列鉴定正确的重组质粒转化进入表达菌株TransB,诱导重组蛋白表达。用纯化的重组gH蛋白免疫8周龄BALB/c雄鼠,经杂交瘤技术获得5株稳定分泌抗gH单克隆抗体的细胞株,分别命名为8A9、8B4、8C4、8D9、8D12。所获5株单克隆抗体对gH蛋白均具有良好的反应性,其中8A9、8B4、8D9和8D12具有一定的病毒捕获能力,且8D9和8D12的捕获能力较强。本研究获得了具有自主知识产权的抗gH单克隆抗体,抗体具有良好的病毒捕获能力。在此基础上,我们将进一步鉴定其是否为中和抗体,为今后开发治疗HCMV感染的中和抗体奠定基础。     

神经胶质瘤细胞ATF5表达水平对HCMV复制的影响

人巨细胞病毒(human cytomegalovirus,HCMV)在神经胶质瘤细胞中的复制水平不一,其机制尚不清楚。本研究通过下调转录激活因子5(ATF5)在神经胶质瘤细胞中的表达,检测HCMV感染神经胶质瘤细胞后病毒复制水平的变化。首先用HCMV AD169(MOI=5)分别感染U87、SY5Y及A172细胞,观察细胞形态变化,分别在24、48、72、96、120 h取各时间点上清液检测病毒滴度;Real-time PCR检测HCMV即刻早期基因(IE2)、早期基因(UL44)、晚期基因(UL99)及ATF5的表达情况;Western-blot检测病毒基因编码蛋白及ATF5表达的情况。结果显示HCMV在U87、SY5Y细胞中复制水平与病毒在A172细胞中复制水平相比,U87、SY5Y细胞组明显高于A172细胞组(P0.05),ATF5表达在U87、SY5Y细胞组与A172细胞组相比,U87、SY5Y细胞组ATF5表达明显高于A172组(P0.05);利用慢病毒介导的RNA干扰技术下调ATF5在U87、SY5Y细胞的表达,用HCMV感染细胞检测病毒基因及蛋白的表达,结果ATF5表达下调可抑制HCMV的复制(P0.05)。以上结果表明,在胶质瘤细胞中下调ATF5表达水平可以抑制HCMV的复制水平。     

BrdU标记的人巨细胞病毒感染HEL细胞的方法学研究

目的 以标记在人巨细胞病毒(HCMV)DNA上的BrdU为示踪剂,研究病毒在受染HEL细胞中的移动过程;同时结合病毒蛋白pp65的表达探讨病毒复制、增殖的过程。方法 以BrdU标记的HCMV(MOI=4)感染HEL细胞,分别选取感染后2h、4h、6h、24h及48h 5个时间点的细胞,用抗BrdU单克隆抗体,研究病毒核酸的胞内定位;同时用抗HCMV蛋白pp65的单克隆抗体检测此蛋白的表达及分布。结果 免疫细胞荧光染色结果提示:在感染5个时间点,病毒DNA依次位于胞质、胞核及同时位于胞核和胞质;蛋白pp65的表达及分布规律为:胞内无表达、胞核分布、胞核与胞质同时分布及巨细胞和融合细胞内分布。结论 以BrdU为标记物标记双链DNA病毒核酸不仅为研究HCMV．的胞内移动提供了良好的模型,同时也为其他病毒的研究提供了良好的工具;本实验结合HCMV蛋白pp65的表达和分布直观地反应了HCMV感染HEL细胞并在其中复制、增殖的过程。     

人巨细胞病毒miR-UL22A的重组载体构建以及表达分析

优化构建人巨细胞病毒(human cytomegalovirus,HCMV)miR-UL22A的真核表达载体;明确HCMV临床株及实验室株感染过程中miR-UL22A-5p及miR-UL22A-3p的表达情况。扩增HCMV UL22A编码区不同长度(975、494、209及140 bp)的序列,构建表达miR-UL22A的重组p Silencer(p S)载体,转染人胚肾293细胞后提取总RNA;收集HCMV临床株Han株以及实验室株Towne株感染人胚肺成纤维细胞后6、12、24、48和72h以及不同感染时相(即刻早期、早期、晚期)的总RNA标本;应用Taq Man颈环-实时荧光定量PCR技术检测上述标本miR-UL22A-5p及miR-UL22A-3p的表达量。成功构建了包含上述不同长度miR-UL22A编码序列的重组载体;侧翼序列长度各为40 bp左右的pre-miRNA(即插入140 bp的编码序列)表达成熟miRNA的效果最佳;在病毒的一个复制周期内,HCMV临床株和实验室株miR-UL22A的表达趋势无明显差异,均在感染后6h即检测到表达,72 h达到峰值,且在即刻早期即有miRNA的明确表达;无论是在重组载体表达状态下还是自然感染状态下,miR-UL22A-5p始终为miR-UL22A前体优势表达的miRNA。结果表明,有140 bp片段miRUL22A编码序列的重组载体能够高效地异源表达miR-UL22A-5p及miR-UL22A-3p;miR-UL22A-5p始终为miR-UL22A前体优势表达的miRNA。为进一步研究miRNA转录后调控作用提供了参考。     

人巨细胞病毒感染通过Wnt/β-catenin通路抑制滋养细胞的增殖及侵袭

人巨细胞病毒(Human cytomegalovirus,HCMV)感染能够影响胎盘绒毛外滋养细胞的增殖、侵袭,进而参与病理妊娠的发生,但具体的机制尚未阐明.在真皮成纤维细胞中的研究表明,HCMV能够抑制Wnt/β-catenin通路.因此,为了观察HCMV感染通过Wnt/β-catenin通路抑制滋养细胞增殖及侵袭的作用,本研究培养了人绒毛外滋养细胞株HTR8/SVneo.研究分为对照组、HCMV组、HCMV+LiCl组细胞.对照组细胞用培养液处理,HCMV组细胞用含有3个感染复数(Multiplicity of infection,MOI)HCMV的培养液处理,HCMV+LiCl组细胞用含有3MOI HCMV及50mmol/L Wnt/β-catenin通路激活剂LiCl的培养液处理.48小时后,采用MTS法检测细胞增殖,采用Transwell检测细胞侵袭,采用western blot检测细胞周期蛋白D1(cyclin D1)、基质金属蛋白酶2(Matrix metalloproteinase 2,MMP2)、基质金属蛋白酶9(Matrix metalloproteinase 9,MMP9)、β-连环蛋白(β-catenin)和磷酸型糖原合酶激酶-3β(p-GSK-3β)的表达量.结果显示,与对照组细胞相比,HCMV组细胞的O.D490水平、侵袭数目及cyyclinD1、MMP2、MMP9、β-catenin、p-GSK-3β的表达量均降低(P＜0.05);与HCMV组细胞比较,HCMV+LiCl组细胞的O.D490水平、侵袭数目及cyclinD1、MMP2、MMP9、β-catenin、p-GSK-3β的表达量均增加(P＜0.05).以上结果表明,HCMV感染抑制人绒毛外滋养细胞的增殖和侵袭,且该作用与抑制Wnt/β-catenin通路有关.本研究阐明了HCMV感染通过抑制Wnt/β-catenin信号通路的激活而抑制滋养细胞增殖及侵袭的分子机制,为最终阐明HCMV先天感染的致病机理积累了有价值的研究资料.     

NDV CN株对几种人肿瘤细胞的杀伤作用及其机理的分析

本文研究了NDV-CN株对5株不同的人肿瘤细胞的体外杀伤作用.结果表明5株肿瘤细胞对NDV-CN敏感,于感染早期出现细胞收缩变圆,失去贴壁性,感染第5天时细胞存活率低于10%.其中尤以HEP3B细胞最敏感.但NDV-CN株对人二倍体细胞2BS有弱杀伤性.病毒感染早期可检测到感染细胞中有病毒核酸的复制、感染细胞表面有病毒蛋白的表达,胞浆内有病毒粒子存在.NDV-CN株主要诱导HEP3B及T24细胞产生凋亡,主要诱导Hep2、Hela及A549细胞产生坏死.     

喜树碱对人类巨细胞病毒诱发染色体畸变的频率的调节效应

本文评价了所选用的DNA修复抑制剂对人类巨细胞病毒(HCMV)诱发外周血淋巴细胞(PBLs)染色体畸变频率的影响。以拓扑酶Ⅰ的一种抑制剂——喜树碱(0.05—0.3μg/m1)处理HCMV感染的人类PBLs30小时,结果导致HCMV诱发的染色体损伤频率显著的协同性增加(P<0.O1)。另一方面以ADP核糖聚合酶的一种抑制剂——3—氨基苯酰胺(3—AB)(3—30μg/ml),或者拓扑酶Ⅱ的一种抑制剂——新霉素(3—30μg/ml)处理HCMV感染的PBLs30时小,染色体损伤频率未见明显增加。在喜树碱处理的HCMV感染细胞中,染色单体型断裂包括染色体交换是染色体畸变的主要类型,这提示HCMV感染与单链NDA断裂有关,这些发现还提示,HCMV感染不会造成通过3-AB或新霉素敏感途径修复的直接DNA损伤。     

HCMV感染对恶性胶质瘤U87细胞增殖及ATF5表达的影响

人巨细胞病毒(HCMV)能够诱导肿瘤细胞的恶性转化,但其分子机制尚有待进一步探索。探讨HCMV是否通过调控转录激活因子5(ATF5)的表达变化促进胶质瘤细胞的增殖。采用HCMV AD169株(MOI=5)感染神经胶质瘤U87细胞株,MTT方法观察HCMV感染0、12、24、48 h后细胞的增殖活性。Real-time PCR及Western-blot检测HCMV感染U87细胞后ATF5基因及蛋白的表达水平变化。以慢病毒为载体的靶向ATF5小干扰RNA构建载体,敲低ATF5表达水平后感染HCMV,MTT检测病毒感染细胞的增殖活性变化。HCMV感染神经胶质瘤U87细胞后,与未感染组比较,增值活性明显升高(P0.05),ATF5表达水平上升,表明HCMV感染使胶质瘤细胞增殖活性提高,细胞抗凋亡能力增强。成功构建沉默ATF5细胞系siATF5 U87,HCMV感染siATF5 U87细胞后使细胞增殖活性减弱,抗凋亡能力下降。以上实验结果表明,HCMV感染上调胶质瘤U87细胞ATF5的表达水平,促进细胞的增殖。因此HCMV感染可能通过调控ATF5信号通路增加细胞恶性性状,为治疗胶质瘤提供一个新的思路。     

粉纹夜蛾细胞系QB—Tn9-4s的克隆以及克隆株生物学特性的研究

对粉纹夜蛾Trichoplusia ni细胞系QB-Tn9-4s进行细胞克隆,获得了8个细胞克降株,分别命名为QB-Tn-A、B、C、D、E、F、G和H.对基因组DNA进行RAPD-PCR鉴定,各细胞克隆株与原始细胞系具有相同的DNA扩增谱带.各细胞克隆株在形态和生物学特性方面表现出一定的差异.克隆株QB-Tn-A、B、c、D和E以梭形细胞为主,大约占细胞总数的60%～80%;F、G和H以棒状细胞为主,比例分别为44.5%、49.5%和80.O%.8个克隆株对苜蓿银纹夜蛾核型多角体病毒(AcMNPV)均较敏感,感染率均在92%以上,平均每个细胞病毒多角体(OBs)产量在78～110个之间,其中克隆株QB-Tn-A多角体产量最高达110个,略高于BTI-Tn5Bl-4和QB-Tn9-4s,明显高于Sf-9细胞;克隆株QB-Tn-E、H、A和C的fIJ芽性病毒(BV)产量与原始细胞系(3.37×107TCID50/mL)接近,而其它4株均低于原始细胞系.     

2003年国际生物学奥林匹克竞赛理论试题答案

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A human cytomegalovirus function inhibits replication of herpes simplex virus.

Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 micrograms/ml) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5 degrees C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.     

Expression of human cytomegalovirus immediate early antigens is responsible for a novel chromatin structure of infected human embryonic lung cells

Whereas human embryonic lung (HEL) cells displayed chromatin fibers composed of a repeat of conventional nucleosomes of 15 nm in diameter, human cytomegalovirus (HCMV) infection induced transient appearance of a novel chromatin structure composed of a repeat of large ellipsoids of 45-65 nm X 15-30 nm with linkers of 50-60 nm long and 6-7 nm thick. Essentially the same change in chromatin structure could be induced when uninfected HEL cell nuclei were incubated in vitro with a 0.4 M NaCl nuclear extract from HCMV-infected HEL cells expressing immediate early antigens (IEA's) or with a similar nuclear extract from NIH/3T3 cells constitutively expressing HCMV IEA's. The latter cell line was established by transformation of the mouse cells with a plasmid carrying the HCMV major immediate early and immediate early 2 genes. These results together with those of control experiments suggest that the expression of IEA's is directly or indirectly responsible for the appearance of the novel chromatin structure in HCMV-infected HEL cells.     

Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest.

Human cytomegalovirus (HCMV) infection stimulates cellular DNA synthesis and causes chromosomal damage. Because such events likely affect cellular proliferation, we investigated the impact of HCMV infection on key components of the cell cycle. Early after infection, HCMV induced elevated levels of cyclin E, cyclin E-associated kinase activity, and two tumor suppressor proteins, p53 and the retinoblastoma gene product (Rb). The steady-state concentration of Rb continued to rise throughout the infection, with most of the protein remaining in the highly phosphorylated form. At early times, HCMV infection also induced cyclin B accumulation, which was associated with a significant increase in mitosis-promoting factor activity as the infection progresses. In contrast, the levels of cyclin A and cyclin A-associated kinase activity increased only at late times in the infection, and the kinetics were delayed relative to those for cyclins E and B. Analysis of the cellular DNA content in the infected cells by flow cytometry showed a progressive shift of the cells from the G1 to the S and G2/M phases of the cell cycle, leading to an accumulation of aneuploid cells at late times. We propose that these HCMV-mediated perturbations result in cell cycle arrest in G2/M.     

The effects of cytomegalovirus on human immunodeficiency virus replication in brain-derived cells correlate with permissiveness of the cells for each virus.

Human cytomegalovirus (HCMV) is commonly found in the brains of patients with AIDS and in some cases can be detected in the same cells as can human immunodeficiency virus type 1 (HIV-1). In this study, we analyzed the patterns of replication of HIV-1 and HCMV in singly infected cells and the effects of dual infection in human brain-derived cell lines of three different origins: neuroblastoma cell lines SK-N-MC and SY5Y; astrocytoma/glioblastoma cell lines U373-MG and Hs 683; and undifferentiated glioblastoma cell lines A172 and T98G. To bypass the restriction at the adsorption/penetration step in these CD4-negative cells, we used HIV-1 (amphotropic retrovirus) pseudotypes. These HIV-1 pseudotypes infected the majority of the cells in the cultures and expressed high levels of HIV-1 gene products in all except the SY5Y cells. The cell lines differed in the ability to support HCMV infection, but coinfection with HIV-1 had no effect on HCMV replication. The A172 cells were completely nonpermissive for HCMV gene expression, while HCMV replication in the singly infected T98G and SK-N-MC cell lines was restricted at the level of some early gene products. This resulted in complete and partial inhibition, respectively, of viral DNA synthesis. Dual infection of the A172, T98G, and SK-N-MC cells had no effect on HIV-1 replication. The other three cell lines, U373-MG, Hs 683, and SY5Y, were fully permissive for HCMV replication. In the U373-MG and Hs 683 cells, HCMV markedly inhibited the synthesis of HIV-1 gene products. In contrast, a transient stimulation of HIV-1 production followed by a repression was observed in the dually infected SY5Y cells. We conclude from these results that under conditions in which both HIV-1 and HCMV can undergo fully permissive infection, HCMV can repress HIV-1 gene expression. In cells in which HCMV replication is limited but HIV-1 replicates well, there is no effect on HIV-1 gene expression. However, activation of HIV-1, at least transiently, may occur in cells in which HIV-1 gene expression is limited. These studies suggest that a threshold level of some HIV-1 gene product(s) may obscure activation or promote repression of HIV replication by HCMV.     

Constitutively active Gq/11-coupled receptors enable signaling by co-expressed G(i/o)-coupled receptors

Co-expression of guanine nucleotide-binding regulatory (G) protein-coupled receptors (GPCRs), such as the G(i/o)-coupled human 5-hydroxytryptamine receptor 1B (5-HT(1B)R), with the G(q/11)-coupled human histamine 1 receptor (H1R) results in an overall increase in agonist-independent signaling, which can be augmented by 5-HT(1B)R agonists and inhibited by a selective inverse 5-HT(1B)R agonist. Interestingly, inverse H1R agonists inhibit constitutively H1R-mediated as well as 5-HT(1B)R agonist-induced signaling in cells co-expressing both receptors. This phenomenon is not solely characteristic of 5-HT(1B)R; it is also evident with muscarinic M2 and adenosine A1 receptors and is mimicked by mastoparan-7, an activator of G(i/o) proteins, or by over-expression of Gbetagamma subunits. Likewise, expression of the G(q/11)-coupled human cytomegalovirus (HCMV)-encoded chemokine receptor US28 unmasks a functional coupling of G(i/o)-coupled CCR1 receptors that is mediated via the constitutive activity of receptor US28. Consequently, constitutively active G(q/11)-coupled receptors, such as the H1R and HCMV-encoded chemokine receptor US28, constitute a regulatory switch for signal transduction by G(i/o)-coupled receptors, which may have profound implications in understanding the role of both constitutive GPCR activity and GPCR cross-talk in physiology as well as in the observed pathophysiology upon HCMV infection.     

Presentation of type B peptide-MHC complexes from hen egg white lysozyme by TLR ligands and type I IFNs independent of H2-DM regulation

In APCs, presentation by MHC II molecules of the chemically dominant peptide from the protein hen egg white lysozyme (HEL) generates different conformational isomers of the peptide-MHC II complexes (pMHC). Type B pMHCs are formed in early endosomes from exogenous peptides in the absence of H2-DM, whereas in contrast, type A pMHC complexes are formed from HEL protein in late vesicles after editing by H2-DM. Thus, H2-DM edits off the more unstable pMHC complexes, which are not presented from HEL. In this study, we show that type B pMHC complexes were presented from HEL protein only after stimulation of dendritic cells (DC) with TLR ligands or type I IFN. Type I IFN contributed to most TLR ligand-induced type B pMHC generation, as presentation decreased in DC lacking the receptor for type I IFNs (IFNAR1(-/-)). In contrast, presentation of type A pMHC from HEL and from peptide was minimally affected by TLR ligands. The relative effectiveness of CD8α(+) DC or CD8α(-) DC in presenting type B pMHC complexes varied depending on the TLR ligand used. The mechanisms of generation of type B pMHC from HEL protein with TLR stimulation did not involve H2-DM or release of peptides. DC from H2-DM-deficient mice in the presence of TLR ligands presented type B pMHC. Such DC showed a slight enhancement of HEL catabolism, but peptide release was not evident. Thus, TLR ligands and type I IFN alter the pathways of presentation by MHC II molecules of DC such that type B pMHCs are generated from protein Ag.     

The switch from latent to productive infection in epstein-barr virus-infected B cells is associated with sensitization to NK cell killing

Following activation of Epstein-Barr virus (EBV)-infected B cells from latent to productive (lytic) infection, there is a concomitant reduction in the level of cell surface major histocompatibility complex (MHC) class I molecules and an impaired antigen-presenting function that may facilitate evasion from EBV-specific CD8+ cytotoxic T cells. In some other herpesviruses studied, most notably human cytomegalovirus (HCMV), evasion of virus-specific CD8+ effector responses via downregulation of surface MHC class I molecules is supplemented with specific mechanisms for evading NK cells. We now report that EBV differs from HCMV in this respect. While latently infected EBV-positive B cells were resistant to lysis by two NK lines and by primary polyclonal NK cells from peripheral blood, these effectors efficiently killed cells activated into the lytic cycle. Susceptibility to NK lysis coincided not only with downregulation of HLA-A, -B, and -C molecules that bind to the KIR family of inhibitory receptors on NK cells but also with downregulation of HLA-E molecules binding the CD94/NKG2A inhibitory receptors. Conversely, ULBP-1 and CD112, ligands for the NK cell-activating receptors NKG2D and DNAM-1, respectively, were elevated. Susceptibility of the virus-producing target cells to NK cell lysis was partially reversed by blocking ULBP-1 or CD112 with specific antibodies. These results highlight a fundamental difference between EBV and HCMV with regards to evasion of innate immunity.