传染性软疣病毒（MCV）的电镜观察

传染性软疣病毒（ＭＣＶ）的电镜观察李德忠,肖同浩,武晓华（广州军区武汉总医院电镜室,武汉４３００７０）吴宁（广州军区武汉总医院皮肤科,武汉４３００７０）关键词传染性软疣病毒,病毒形态,电镜观察曾有研究描述ＭＣＶ的发育周期中有８种形态,也有按发育过程将...     

人巨细胞病毒转化作用的研究进展

人巨细胞病毒（ＨＣＭＶ）属于ＤＮＡ肿瘤病毒,有３个可导致细胞恶性转化的形态转化区,另有病毒即刻早期（ＩＥ）基因的编码产物对细胞生长进行调探。本文最后探讨了病毒导致细胞恶性转化的可能机制。     

血沉标本中人巨细胞病毒DNA的检出

用聚合酶链反应（ＰＣＲ）和地高辛标记探针（ｄｉｇ－ｐｒｏｂｅ）检测临床血沉标本中人巨细胞病毒（ＨＣＭＶ）ＤＮＡ。结果表明,人群血标本中ＨＣＭＶ－ＤＮＡ携带率较高;ＰＣＲ技术较ｄｉｇ－ｐｒｏｂｅ更敏感、快速、简便,二者检出ＨＣＭＶ－ＤＮＡ阳性率分别为８３．３％和６０．３％;ＨＣＭＶ－ＤＮＡ检出率、ＨＣＭＶ－ＩｇＭ检出率与血沉值高低之间无相关性。     

杆状病毒DNA解旋酶

ＤＮＡ解旋酶 (ｈｅｌｉｃａｓｅ)是ＤＮＡ复制过程中一类重要的酶 ,负责打开ＤＮＡ双链 ,并参与新生链的合成 ,在ＤＮＡ修复和重组过程中都发挥着必不可少的作用。杆状病毒ＤＮＡ解旋酶除了参与ＤＮＡ复制外 ,对于晚期基因的转录、关闭宿主蛋白质合成及决定杆状病毒宿主域方面都有重要的作用。1.杆状病毒解旋酶的结构目前已有 5种杆状病毒的ＤＮＡ解旋酶基因得到克隆并测序 ,分别是苜蓿银纹夜蛾核多角体病毒(ＡｃＭＮＰＶ) ,黄杉毒蛾核多角体病毒 (ＯｐＭＮＰＶ) ,家蚕核多角体病毒 (ＢｍＮＰＶ) ,甜菜夜蛾核多角体病毒(ＳｅＭＮＰＶ)及粉…     

人免疫缺陷病毒I型抗AZT抗药株的体外研究

人免疫缺陷病毒Ⅰ型（ＨＩＶ－１）抗３＇─叠氮─３＇─脱氧胸腺嘧啶（ＡＺＴ）抗药株经体外感染Ｃ８１６６淋巴细胞在高浓度ＡＺＴ条件下筛选获得,并暂命名为ＨＩＶ─１─Ｒ株。该抗药株与ＨＴＬＶ─ⅢＢ株相比,在同一感染复数（Ｍ０１）病毒量感染Ｃ８１６６细胞,经不同浓度的ＡＺＴ处理后,其复制的病毒量和对ＡＺＴ的敏感性有显著的差异,抗药株感染Ｃ８１６６细胞,加ＡＺＴ处理后,分离细胞ＤＮＡ作ＰＣＲ扩增后分析,特异性病毒的ＤＮＡ量比敏感株高１００倍以上,显示其抗药性作用点在病毒逆转录ＤＮＡ前。     

人巨细胞病毒的分子克隆及其特异性DNA探针的制备

从人巨细胞病毒（ＨＣＭＶ）培养物中提取ＨＣＭＶ并抽提其ＤＮＡ,经限制性内切酶ＢａｍＨＩ完全消化后,与质粒ｐＢｌｕｅｓｃｒｉｐｔ－ＳＫ重组建立了ＨＣＭＶ的ＤＮＡ文库,从此文库。中随机筛选出两个重组质粒（ｐＣＭＶ－１和ｐＣＭＶ－２）,用ＢａｍＨＩ分析证明其中所含的病毒ＤＮＡ片段的大小分别为１．０ｋｂ和７．５ｋｂ,将这两种质粒大量扩增纯化后,用光生物素进行标记作为探针,证明其只与ＨＣＭＶ反应,与正常人细胞ＤＮＡ及Ⅰ型和Ⅱ型单纯疤疹病毒ＤＮＡ无交叉反应。     

中国棉铃虫核型多角体病毒DNA聚合酶基因的克隆和序列分析

应用谷实夜蛾核型多角体病毒（ＨｚＳＮＰＶ）ＤＮＡ聚合酶基因ＨｉｎｄⅢ／ＰｓｔⅠ３５９６ｂｐ片段作探针,经Ｓｏｕｒｔｈｅｒｎｂｌｏｔ杂交,克隆了中国棉铃虫核型多角体病毒（ＨａＳＮＰＶ）完整的ＤＮＡ聚合酶基因,大小约为３．４ｋｂ。限制性内切酶分析表明,ＨａＳＮＰＶＤＮＡ聚合酶基因限制性内切酶图谱与ＨｚＳＮＰＶ相似。用双脱氧链终止法测定该基因部分核苷酸序列（８０５ｂｐ）,推导出编码区２０６ａ。序列同源性比较显示,ＨａＳＮＰＶＤＮＡ聚合酶与ＨｚＳＮＰＶ之间具有高度的同源性;与ＬｄＭＮＰＶ、ＡｃＭＮＰＶ、ＢｍＳＮＰＶ、ＣｆＭＮＰＶ和ＯｐＭＮＰＶ也具有一定的同源性     

一种中国发生的真菌传小麦花叶病毒RNA－13＇末端核苷酸序列分析

以采自河南的真菌传小麦花叶病毒（ＦＷＭＶ－Ｃ）为材料,抽提病毒ＲＮＡ,合成互补ＤＮＡ（ｃＤＮＡ）。对杂交筛选所得ｃＤＮＡ克隆进行亚克隆及序列分析,结果表明,亚克隆ｐＧＳＩ含有一个长度为８９１个核苷酸的不完整开放阅读框架（ＯＲＦ）和长度为２５８个核苷酸的３＇末端非编码区（ＮＴＲ）,并带有Ｐｏｌｙ（Ａ）尾序。此段序列与大麦黄花叶病毒（ＢａＹＭＶ）及法国报道的一种小麦梭条花叶病毒（ＷＳＳＭＶ－Ｆ）的ＲＮＡ－１３＇末端分别具有６７．６％和６９．９％的同源性。由所测序列编码区（１－８９１ｎｔ）可推导产生２９６个氢基酸,并与ＷＳＳＭＶ－Ｆ及ＢａＹＭＶ外壳蛋白氨基酸序列分别具有７５．９％和７１％的同源性。此结果表明,ＦＷＭＶ－Ｃ为另外一种不同于ＷＳＳＭＶ－Ｆ的大麦黄花叶病毒组（Ｂａｙｍｏｖｉｒｕｓ）病毒,所测基因组片段应为ＲＮＡ－１３＇末端序列,其中可能包括了病毒全长外壳蛋白编码区域。     

Epstein—Barr病毒膜抗原基因免疫的研究

将Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）膜抗原（ＭＡ）ＢＬＬＦ１基因,插入含有ＣＭＶ启动子的真核表达载体ｐｃＤＮＡ３下游ＢａｍＨＩ位点,构建成真核表达质粒ｐｃＤＮＡ３－ＭＡ。将纯化的ＤＮＡ注射Ｂａｌｂ／ｃ小鼠股四头肌。经免疫的动物产生抗ＥＢ病毒ＭＡ特异性的抗体和中和抗体,依赖抗体细胞介导的细胞毒作用（ＡＤＣＣ）,特异性Ｔ淋巴细胞增生性反应及细胞毒性Ｔ淋巴细胞（ＣＴＬ）杀伤作用。基因免疫与基因－蛋白     

单股环DNA病毒基因组的复制及其转录调控因子结合序列

单股环ＤＮＡ病毒基因组的复制及其转录调控因子结合序列崔治中（扬州大学农学院兽医系,生物技术系,扬州２２５００１）关键词单股环ＤＮＡ病毒,基因组复制,调控区结构最近定名的单股环ＤＮＡ病毒科（Ｃｉｒｃｏｖｉｒｉｄａｅ）是迄今为止发现的一类最小的动物病毒。...     

Effect of aluminum chloride and zinc sulfate on Autographa california nuclear polyhedrosis virus (ACNPV) replication in cell culture

When IPL-SF-21AE III continuous insect cell line was grown and maintained in IPL-41 insect cell culture medium supplemented with 16 microM of AlCl3 or 0.24 microM of ZnSO4 . 7H2O, or both metallic salts, and then infected with Autographa california nuclear polyhedrosis virus, virus replication was increased significantly. The yield of polyhedral inclusion bodies (PIB) was enhanced up to 121%. Synthesis of cell-free nonoccluded virus was increased to 365% when infectivity was assayed by the plaque method. Newly applied electron microscopic quantitation and stereological techniques also revealed a significant increase in virus particles (VP) and in amount and size of PIB as well as number of VP per PIB.     

Development of a novel expression vector system using Spodoptera exigua nucleopolyhedrovirus

To develop a novel Spodoptera exigua nucleopolyhedrovirus (SeNPV) expression vector system, we examined characteristics of the SeNPV polyhedrin expression in S. exigua cells (Se301). While the extracellular virus titer of SeNPV was 100-fold lower than that of Autographa californica nucleopolyhedrovirus (AcNPV), the levels of polyhedral inclusion body (PIB) formation and polyhedrin expression were higher in SeNPV. To investigate foreign gene expression under the control of the polyhedrin promoter, polyhedrin-based transfer vector pSeKSK2 was constructed, and then recombinant virus SeK1-LacZ was constructed by inserting E. coli lacZ gene as a reporter gene into a genomic DNA of SeNPV using this transfer vector. The beta-Galactosidase activity of SeK1-LacZ in Se301 was about 1.3 times higher than that of BacPAK6. Thus, the SeNPV expression vector system constructed in this study would be very useful in the expression of foreign proteins, specifically for the enhancement of the pesticidal properties of SeNPV by inserting pesticidal genes.     

Molecular cloning of the avian myelocytomatosis virus genome and recovery of infectious virus by transfection of chicken cells.

The avian retrovirus myelocytomatosis virus 19 (MCV) possesses an interesting diversity of oncogenic potentials, but the virus has proven difficult to study because of its inability to replicate without the assistance of a helper virus. We have therefore isolated and amplified the genome of MCV by molecular cloning in a procaryotic vector. The topography of the cloned DNA was explored by the use of restriction endonucleases and radioactive complementary DNAs representing specific domains in avian retrovirus genomes. The cloned DNA appeared to be an authentic representation of the MCV genome: the size and genetic topography of the DNA were comparable to those of MCV, and transfection of the cloned DNA into chicken cells (in company with the DNA of a suitable helper virus) gave rise to virus with the genome and transforming potentials of MCV. The availability of cloned MCV DNA should facilitate a variety of genetic and biochemical manipulations directed at elucidating the mechanism of oncogenesis by MCV.     

Influence of larval stage and virus inoculum on virus yield in insect host Neodiprion abietis (Hymenoptera: Diprionidae)

Virus yield produced by dead larvae of balsam fir sawfly, Neodiprion abietis (Harris) (Hymenoptera: Diprionidae), that had been infected at four different larval stages (second, third, fourth, or fifth instar) with two virus concentrations (10(5) polyhedral inclusion bodies (PIB) /ml or 10(7) PIB/ml), were analyzed and compared to determine the effects of instar and amount of virus inoculum on virus production. The results indicate that both larval stage and inoculation dosage significantly affect virus yield. On average, each dead larva produced 1.36-12.21 x 10(7) PIB, depending upon larval age and virus concentration of inoculation. Although each dead larva produced more PIB when it was inoculated in the fourth or fifth stage, inoculation of these larvae did not result in the highest virus yield because of low larval mortality. In terms of net virus return, third instars would maximize virus yield when they are inoculated with a virus concentration that can cause 95-100% larval mortality.     

Structural studies on the polyhedral inclusion bodies, virions, and DNA of the nuclear polyhedrosis virus of the cotton bollworm Heliothis zea.

The polyhedral inclusion body of the cotton bollworm nuclear polyhedrosis virus contains virions occluded in an orthogonal crystalline matrix. The virions appear as rods or, more frequently, as oval structures that form upon bending of the nucleocapsid within the viral membrane. The nucleocapsid consists at least of DNA surrounded by a capsid composed of subunits, possibly helically arranged. The viral DNA is circular and supercoiled. It is heterogenous in size with contour lengths ranging from 15 to 45 mum.     

Yield and activity of theHeliothis zea single nuclear polyhedrosis virus propagated in cloned and uncloned lines ofHeliothis cells

Summary Heliothis cell lines originated from different laboratories were characterized by isoenzyme analysis and then evaluated for their ability to produce the single nuclear polyhedrosis virus ofHeliothis zea (HzSNPV). A cloned cell line (designated Hzlb3), whose homogeneity was supported by both morphological and isoenzyme analysis, was derived from a parental line (Hzl). Significantly greater yields (about 10-fold) of tissue-culture-derived, non-occluded virus (TCNOV) were obtained when compared to the parental line. The Hzlb3 clone also gave significantly higher yields of TCNOV than the Hz3 and UND-K cell lines. Although lines Hzl, Hz3, and Hzlb3 produced significantly more polyhedral inclusion bodies (PIB) than line UND-K, the infectivity of PIB from UND-K equaled that of lines Hzl and Hzlb3.     

Crystal structure of a bacterial type IB DNA topoisomerase reveals a preassembled active site in the absence of DNA

Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-A crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) beta-sheet domain (amino acids 1-90) and a predominantly alpha-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an "open" conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.     

Mutational biases associated with potential iron-binding DNA motifs in rodent lacI and human p53 mutational databases

The role of Fenton oxidants in DNA damage, aging, and cancer is appreciated, but not well understood. Six potential iron-binding (PIB) DNA motifs were previously identified as sites of preferential strand cleavage. Since DNA-metal binding domains are a known determinant of oxidative DNA damage, and the location of strand breaks explains where oxidant attack occurs, we sought to determine whether the likelihood of base change mutations is a function of neighboring PIB motifs. We developed a sliding window function that computes the density of PIB motifs on both strands, within 4-12bp, for each location along a target gene. This range of window sizes reflects known diffusion distances of Fenton reaction products. Using mutational databases, odds of mutation at each base were calculated relative to PIB motif density, for all PIB motif types in aggregate, or for individual PIB motifs. Using mutational data from lacI transgenic animals, we observed a non-random distribution of PIB motifs, associated with increased odds of mutation, showing a strand bias. Sensitivity analysis confirmed that the optimum association between PIB motif density and mutations occurs when a 7bp radius is used for the window size. Randomly simulated mutations showed no association with PIB motif density. When the method was applied to human TP53 mutation data, we saw similar results, but no strand bias. As PIB motif density rises, linear trends are observed for increasing odds of mutation. Sensitivity analysis revealed associations between PIB motifs and GC --> AT transitions and GC --> TA transversions-the most commonly observed types of mutations arising from oxidative DNA damage. DNA-metal binding motifs are found in a wide variety of biological contexts, including many where conformational sensitivity to redox state is important. These techniques can help elucidate how DNA-iron-binding may affect lesions and subsequent mutations from multiple agents.     

HIV-1 Gag蛋白在大肠杆菌表达系统中诱导和纯化条件的优化

为探讨诱导温度对于HIV-1 Gag在大肠杆菌中表达产物状态以及尿素浓度对蛋白纯化效果的影响, 将30oC和37oC诱导表达的包涵体分别溶于不同浓度的尿素, 比较溶解性的差异, 并比较复性的不同。将30oC诱导的目的蛋白分别用2 mol/L和8 mol/L尿素溶解后做层析分离, 比较两者的分离效果。结果发现, 与37oC相比, 30oC诱导表达的蛋白能有效溶于低浓度尿素, 并且更容易复性。与8 mol/L尿素溶解相比, 30oC诱导的包涵体用2 mol/L尿素溶解后通过凝胶过滤和离子交换层析纯化能得到更好的分离效果。这提示低温诱导的Gag包涵体中可能含有更多类似天然态构象的蛋白, 而低浓度尿素有利于保持包涵体中蛋白的天然态构象。从而为包涵体蛋白的诱导表达和分离纯化提供了参考。