鱼类细胞系在病毒学研究中的应用进展

鱼类细胞培养尽管起步较晚,但截至目前已有近280余株不同的鱼类细胞系相继建立起来,在生理学、病毒学、毒理学、肿瘤及基因工程等多个领域发挥重要作用。主要对鱼类细胞系在病毒学研究中的最新应用,并结合作者自身的研究,尤其侧重于鱼类病毒分离、重要功能基因鉴定、抗病毒免疫和病毒致病机理研究等方面的进展作一概述。     

Fortified Sera and Their Use in Environmental Virology

Four commercially available fortified sera were compared to fetal bovine serum (FBS) with regard to their ability to maintain or increase the sensitivity of the Buffalo green monkey (BGM) kidney cell line to viral infection. Nine virus strains and five wastewater samples were used. Fortified sera were comparable to FBS for the enumeration of some viruses by the plaque method and for the detection of virus in wastewater by the most-probable-number assay.     

Development of a Markerless Genetic Exchange System for Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3′)-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.The anaerobic sulfate-reducing bacteria (SRB) are found in a remarkable variety of habitats. These bacteria have received attention recently because they have a potential role in toxic metal bioremediation (23, 26). To fully understand the potential benefits and to maximize opportunities for successful manipulation of the SRB, it would be useful to create deletions in critically important genes. Several activities of particular interest are represented by multiple isozymes, suggesting that compensation may occur upon elimination of one or more of these genes. To fully elucidate alternative pathways, genetic approaches allowing the construction of multiple mutations are needed. The genetic manipulation of the SRB Desulfovibrio vulgaris Hildenborough has seen significant improvements in recent years (reviewed in reference 3). Chloramphenicol and kanamycin marker exchange mutagenesis methods have been developed (2, 10). Although gene deletions can be constructed, the necessary retention of antibiotic resistance limits sequential deletions, since each deletion would require an additional antibiotic cassette. To eliminate the necessity of marker retention, an in-frame markerless deletion system has been developed.A two-step method for marker exchange/deletion that used the counterselectable marker sacB (13) was used by Fu and Voordouw (10) to generate the first deletion by marker exchange in D. vulgaris. The sacB gene from Bacillus subtilis encodes levansucrase and confers sensitivity to sucrose in many gram-negative bacteria (7, 32, 35), including D. vulgaris (8, 10, 15, 18, 19, 21). In the first step of the process, a suicide plasmid carrying DNA regions from up- and downstream of a target gene flanking a chloramphenicol resistance (Cm^r) cassette was introduced into D. vulgaris by conjugation and single recombinants were selected as Cm^r colonies (10). After confirmation of the integration of this plasmid, the double-recombination event was selected on medium containing chloramphenicol and sucrose. This method, with some variation, has been used to make several mutants by the Voordouw group (8, 10, 15, 18, 19, 21). One unexpected complication was the observation that 50% of sucrose resistant colonies were due to events other than the removal of the sacB gene and plasmid through a second recombination as desired (11). Also, sensitivity to sucrose is apparently strongly affected by medium composition, initial culture density, and the time of exposure (11). This method involved a large time investment, but it ultimately resulted in a marker exchange mutant (Cm^r) and established the effectiveness of a two-step recombination process in D. vulgaris.Among alternative counterselectable markers are the purine and pyrimidine salvage enzymes, phosphoribosyl transferases (PRTases). These enzymes allow the recycling of free bases from internal or environmental sources, as well as the incorporation of base analogs into nucleoside monophosphates. Importantly, the incorporation of base analogs can be lethal and are the reason these nucleotide salvage pathways have been widely used as counterselectable markers for gene knockout systems in bacteria, archaea, and eukaryotes (4, 5, 8, 9, 12, 16, 22, 24, 27, 29, 34). Specifically, the incorporation of the pyrimidine analog 5-fluorouracil (5-FU) is lethal in a number of bacteria (8, 16, 22). Mutants whose genes encoding the pertinent PRTases have been deleted are resistant to the toxic base analogs (4, 5, 8, 9, 12, 16, 22, 24, 27, 29, 34). Reintroduction of these genes restores sensitivity. In order to utilize the genes for PRTases as counterselectable markers, a deletion of the endogenous PRTase gene must be created in the host strain. We have previously shown that wild-type D. vulgaris is extremely sensitive to low levels of 5-FU, as little as 0.1 μg/ml (3). In the present study, we deleted the upp gene (DVU1025) encoding the putative uracil phosphoribosyl transferase in D. vulgaris creating strain JW710 and showed that it was resistant to 5-FU. When the upp gene was reintroduced into JW710 (Δupp), it restored sensitivity to wild-type levels of 5-FU. These phenotypic observations indicate that the loss of the upp provides a selectable marker for a two-step integration and excision strategy for the deletion of target genes without a residual marker exchange. A second advantage of using this markerless method is the facile ability to generate in-frame deletions, eliminating potential polarity.To test the effectiveness of using the upp as a counterselectable marker in D. vulgaris, we deleted the gene encoding the endonuclease of a type I restriction-modification system, hsdR (DVU1703), and the downstream conserved hypothetical gene (CHP; DVU1702), creating strain JW7035 [Δupp Δ(hsdR-CHP)]. The type I restriction-modification system was targeted for deletion in hopes of increasing the transformation efficiency of D. vulgaris and facilitating the construction of future deletions. As anticipated, electroporation experiments with stable plasmids revealed an improvement in transformation efficiency for stable plasmids compared to wild-type D. vulgaris. Finally, Gateway Technology (Invitrogen) was applied to generate a destination vector (pMO727) containing the constitutively expressed wild-type upp gene. This vector will expedite the process of creating the required suicide deletion vectors for future markerless deletions.     

多重PCR及其在病毒性疾病诊断中的应用

多重PCR（muhiplex PCR）是在常规PCR基础上改进并发展起来的一种新型PCR技术。该技术可利用多对引物在同一个反应体系中同时扩增多段靶序列,目前广泛应用于科研及临床领域。该文简要介绍多重PCR反应条件的优化及其在病毒感染性疾病诊断中的应用。     

病毒学面临的挑战及其在新世纪发展的机遇

作为微生物学的一个重要分支,病毒学的发展为提高人类健康水平和促进工农业生产做出了突出贡献.我国病毒学虽然在基础研究领域较为薄弱,但在诸如病毒分离、分类和形态研究以及疫苗研制等领域屡有建树,在动植物病毒病的生物防治和人类病毒病以疫苗为主的综合防治方面都做出了具有国际水平的工作.本文尝试从二十世纪医学病毒学的发展出发分析该学科所面临的机遇与挑战,并探讨我国病毒学界应采取的应对策略.     

Evaluation of Gentamicin for Use in Virology and Tissue Culture

Data are presented comparing gentamicin to penicillin and streptomycin (Pen-Strep) in tissue culture medium with respect to a number of parameters associated with virology and tissue culture. Unlike Pen-Strep, gentamicin was stable at pH 2 to 10 for 15 days at 37 C in tissue culture medium, and its activity was unaffected by the presence of serum. Moreover, it was stable to autoclaving. Twenty cell types replicated normally at the suggested concentration of 50 mug/ml, and all cells were unaffected by 20 times this concentration. Evidence for its practical use in virus studies was demonstrated in that (i) it was not viricidal to ribonucleic acid or deoxyribonucleic acid viruses at 40 times the suggested concentration at 37 C, (ii) the size and number of plaques were not affected by 20 times the suggested concentration, (iii) interferon assays and production were unaffected by 20 times the suggested concentrations. Gentamicin may be uniquely useful for shipment of clinical specimens and long-term tissue culture and virus studies.     

A New Method for Use in Plant Virology

Авторы описывают новый метод Фильм с использованием камеры и пленки для изучения формирование и развитие локальных поражений и начало и ход системных инфекции, вызванные различными вирусами растений. Метод до сих пор используется для Вирус табачной мозаики только для изучения местных поражений в Nicotiana glutinosa и системные инфекции в Nicotiana tabacum. Она также может быть использован для изучения других растений вирусов, впрочем, и его преимущество состоит в точной регистрации явлений которые не могут быть установлены на всех, или только очень неточно, по невооруженным глазом. Еще одно преимущество заключается в том, что процесс, один раз сняли, может быть прогнозируемый снова или отдельных местных поражений на различных этапах роста можно проследить на документ по копирование таблицы. Этот метод позволяет точное исследование роста, размеры и Форма местные поражения, которое не было возможности, с методами, используемыми до сих пор.     

Use of a Clonal Line of Porcine Kidney Cell Cultures for Primary Isolation and Vaccine Studies with Adenoviruses

The clonal line (Y15) of porcine kidney stable cells provided a recovery system for adenovirus T4 from specimens from adults with respiratory illnesses that was as sensitive as human embryo kidney cultures. Adenoviruses T7 from adults, and T1, 2, 3, and 5 from children could be readily isolated in porcine kidney cell cultures. The latter were useful for adenovirus vaccine studies in that infectivity titers of live virus vaccine and neutralization antibody responses after vaccination were equal to those obtained in human embryo kidney cultures.     

生物素标记的6种cDNA探针检测S180及其克隆细胞株mRNA的表达

目的检测S180及其克隆细胞株S1B11及S2D9mRNA的表达,对这些细胞株进行识别和质量控制.方法用生物素标记的6种cDNA探针,细胞玻片原位杂交的方法检测细胞中mRNA的表达.结果北京市肿瘤研究所(肿瘤所)保存的S180与生物素标记的P16、c-fos、c-myc及c-jun探针杂交阳性,克隆细胞株S1B11与c-fos及c-jun探针杂交阳性,克隆细胞株S2D9与c-fos、c-myc及c-jun探针杂交阳性.结论肿瘤所S180及其2株克隆细胞中mRNA的表达不同,c-myc基因的表达与否可以把S1B11及S2D9克隆细胞区别开;细胞株致瘤性与癌基因表达有关.     

Whole-Genome Sequence of Corynebacterium pseudotuberculosis Strain Cp162, Isolated from Camel

Corynebacterium pseudotuberculosis is a pathogen of great veterinary and economic importance, since it affects livestock, mainly sheep and goats, worldwide, together with reports of its presence in camels in several Arabic, Asiatic, and East and West African countries, as well as Australia. In this article, we report the genome sequence of Corynebacterium pseudotuberculosis strain Cp162, collected from the external neck abscess of a camel in the United Kingdom.     

Isolation,Characterization of a Strain Capable of Degrading Imazethapyr and Its Use in Degradation of the Herbicide in Soil

One strain of bacterium IM-4, capable of degrading imazethapyr (IMZT), was isolated from the IMZT-contaminated soil. The isolate was identified as Pseudomonas sp. according to its physiological characteristics, biochemical tests, and 16S rRNA gene phylogenetic analysis. This strain could utilize IMZT as the sole carbon and energy source. About 73.4% of the 50 mg l⁻¹ initially added IMZT was degraded after 7 days of inoculation with strain IM-4. This strain also showed the capability to degrade other imidazolinone herbicides such as imazapyr, imazapic, and imazamox. The inoculation strain IM-4 to soil treated with IMZT resulted in a higher degradation rate than in noninoculated soil regardless if the soil was sterilized or nonsterilized. Inoculation of strain IM-4 could also mitigate the phytotoxic effects of IMZT on the growth of maize.     

Isolation and Characterization of a Freeze-Tolerant Diploid Derivative of an Industrial Baker's Yeast Strain and Its Use in Frozen Doughs

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.     

Development of Eimeria meleagrimitis Tyzzer from Sporozoites and Merozoites in Turkey Kidney Cell Cultures*

SYNOPSIS. Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P < 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P < 0.05) than their in vivo counter parts.     

Toxoplasma gondii Development of Its Replicative Niche: in Its Host Cell and Beyond

Intracellular pathogens can replicate efficiently only after they manipulate and modify their host cells to create an environment conducive to replication. While diverse cellular pathways are targeted by different pathogens, metabolism, membrane and cytoskeletal architecture formation, and cell death are the three primary cellular processes that are modified by infections. Toxoplasma gondii is an obligate intracellular protozoan that infects ∼30% of the world''s population and causes severe and life-threatening disease in developing fetuses, in immune-comprised patients, and in certain otherwise healthy individuals who are primarily found in South America. The high prevalence of Toxoplasma in humans is in large part a result of its ability to modulate these three host cell processes. Here, we highlight recent work defining the mechanisms by which Toxoplasma interacts with these processes. In addition, we hypothesize why some processes are modified not only in the infected host cell but also in neighboring uninfected cells.     

Biodegradation of Chlorpyrifos by Enterobacter Strain B-14 and Its Use in Bioremediation of Contaminated Soils

Six chlorpyrifos-degrading bacteria were isolated from an Australian soil and compared by biochemical and molecular methods. The isolates were indistinguishable, and one (strain B-14) was selected for further analysis. This strain showed greatest similarity to members of the order Enterobacteriales and was closest to members of the Enterobacter asburiae group. The ability of the strain to mineralize chlorpyrifos was investigated under different culture conditions, and the strain utilized chlorpyrifos as the sole source of carbon and phosphorus. Studies with ring or uniformly labeled [¹⁴C]chlorpyrifos in liquid culture demonstrated that the isolate hydrolyzed chlorpyrifos to diethylthiophospshate (DETP) and 3, 5, 6-trichloro-2-pyridinol, and utilized DETP for growth and energy. The isolate was found to possess mono- and diphosphatase activities along with a phosphotriesterase activity. Addition of other sources of carbon (glucose and succinate) resulted in slowing down of the initial rate of degradation of chlorpyrifos. The isolate degraded the DETP-containing organophosphates parathion, diazinon, coumaphos, and isazofos when provided as the sole source of carbon and phosphorus, but not fenamiphos, fonofos, ethoprop, and cadusafos, which have different side chains. Studies of the molecular basis of degradation suggested that the degrading ability could be polygenic and chromosome based. Further studies revealed that the strain possessed a novel phosphotriesterase enzyme system, as the gene coding for this enzyme had a different sequence from the widely studied organophosphate-degrading gene (opd). The addition of strain B-14 (10⁶ cells g⁻¹) to soil with a low indigenous population of chlorpyrifos-degrading bacteria treated with 35 mg of chlorpyrifos kg⁻¹ resulted in a higher degradation rate than was observed in noninoculated soils. These results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment.     

PET 微孔编织结构张力环境下诱导细胞迁移机制的 研究现状及发展动态

随着关节韧带损伤发生率日益提高,人工材料植入重建韧带功能是重要治疗手段之一。植入后宿主细胞的趋向、黏附和迁移是人工材料在体内形成长期稳定生物连接的关键。以往研究集中于提高材料生物性,如增加材料亲水性、表面修饰、黏附适宜细胞以及复合细胞因子等,目前在张力环境下三维微孔结构对宿主细胞黏附、迁移、长入和分化影响的研究较少。本文拟以生物相容性良好的聚对苯二甲酸丁二醇酯(PET)材料作为载体,探讨在张力状态下新编PET的结构、孔径大小和孔隙率对细胞迁移等细胞生物学行为影响的研究现状和未来发展。为这些研究的深入指明方向,也为PET作为人工材料修复韧带损伤的临床应用奠定实验基础。     

一株絮凝剂产生菌的筛选及其絮凝特性研究

目的:筛选并研究对有毒物质有一定耐受性的絮凝剂产生菌。方法:利用含苯酚、邻苯二甲酸二丁酯和Pb2(SO4)3的分离培养基从土壤和活性污泥中分离筛选絮凝剂产生菌,对所得的菌种进行摇瓶发酵试验,分别考察其产絮凝剂的周期、絮凝活性分布以及对有毒物质的耐受性等特征,通过提取絮凝剂,将其絮凝活性与其它絮凝剂进行比较。结果:得到一株对苯酚具有一定耐受性的絮凝剂产生菌B2(Serratiasp.),其产絮凝剂的最佳培养时间为48h,絮凝率高于80%。苯酚浓度达0.6g/L时,B2菌的絮凝活性仍高于70%。其90%的絮凝物质集中于菌体,且热稳定性好,对多种悬浊液的絮凝活性高于硫酸铝、PAC。结论:新型絮凝剂产生菌B2对苯酚耐受性强,且絮凝剂提取简便,具有重要的研究价值。