猪水疱病病毒致病和免疫的分子基础

猪水泡病病毒(Swine vesicular disease virus,SVDV)属于小RNA病毒科(Picornaviridae)肠道病毒属(Enterovirus),其抗原特性与柯萨奇病毒B5(Coxsackievirus B5, CVB5)关系密切,被认为是CVB5的一个猪变种或亚种[1]。SVDV只有一个血清型,但对其分离株进行系统发生分析可进一步分为     

猪水疱病病毒致病和免疫的分子基础

猪水泡病病毒(Swine vesicular disease virus,SVDV)属于小RNA病毒科(Picornavirdae)肠道病毒属(Enterovirus),其抗原特性与柯萨奇病毒B5(Coxsackievirus B5,CVB5)关系密切,被认为是CVB5的一个猪变种或亚种[1].SVDV只有一个血清型,但对其分离株进行系统发生分析可进一步分为4个抗原群或基因群[2,3].第1群包含SVDV早期分离株(ITL/1/66),第2群包含1972～1981年间欧洲和日本分离株,第3群包含意大利1988年12月～1992年6月间的分离株,第4群包含1987～1994年间,罗马尼亚、荷兰、意大利和西班牙的分离株.     

猪水泡病病毒VP1基因的克隆和表达

以猪水泡病病毒RNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了849bp的VP1基因,通过T-A克隆技术,将VP1基因片段克隆至pMD18-T克隆载体质粒中,构建SVDV VP1基因克隆重组质粒,进行核苷酸序列分析.然后亚克隆插入pBAD/Thio TOPO表达载体,经测序鉴定,筛选获得VP1基因正向插入、有正确读码框的阳性克隆,成功构建了猪水泡病病毒VP1基因重组表达载体.经L-Arabinose诱导表达,可稳定、高效地表达VP1蛋白抗原.SDS-PAGE结果表明,以终浓度为0.002%的L-阿拉伯醛糖进行诱导,5h后表达可达到高峰,表达蛋白为融合蛋白,质量约47.13kDa,表达产量约占菌体总蛋白的16%.Western blotting检测表明,诱导的蛋白能与猪水泡病阳性血清发生特异性反应.融合蛋白中含有猪水泡病病毒VP1蛋白抗原,为应用该表达蛋白抗原制备SVD免疫血清学诊断试剂和新型疫苗构建奠定基础.     

鸭中人肠道病毒的分离与鉴定

人肠道病毒属于微小核糖核酸病毒科(Picornaviridae)肠道病毒属(Enterovirus)的病毒。其自然宿主主要是灵长目动物。其他动物如牛、猪、鼠、苍蝇、蟑螂等有携带这类病毒的现象,但没有证明它们是自然宿主。文献报道,人的柯萨奇B,和猪的水泡病病原,两者的同源物有50%相同,彼此的抗原性无区别,因此认为人的柯萨奇B,就是猪的水泡病病原。     

稳定表达T7 RNA聚合酶IBRS-2细胞系的建立以及利用该细胞系对SVDV的体内拯救

利用PCR从λ溶原性细菌DE3中扩增出T7 RNA聚合酶基因,定向克隆进逆转录病毒载体pBABEpuro,得到阳性重组质粒pTTBABEpuro.把该重组质粒与pVSV-G共感染GP2-293包装细胞系,形成假型病毒.通过polybrene介导假型病毒感染IBRS-2细胞,利用嘌呤霉素进行传代筛选,形成细胞系IBRST7.对不同代次的IBRST7细胞基因组进行PCR和RT-PCR鉴定,结果表明,T7 RNA聚合酶基因在细胞传代过程中能稳定存在,并能表达目的蛋白的mRNA.为了鉴定T7 RNA聚合酶在IBRST7细胞内是否具有转录活性,扩增口蹄疫病毒(FMDV)的内部核糖体进入位点(IRES)片段和EGFP基因,定向克隆于原核表达载体pET-43.Ia-c( )中,构建了T7启动子控制下转录的具有非帽依赖性表达的重组质粒pIERS-EGFP-ET,把该质粒转染IBRST7细胞,能够在紫外显微镜下观察到绿色荧光,说明EGFP得到了表达,表明IBRST7细胞系内的T7 RNA聚合酶具有转录活性.然后,利用该细胞系成功拯救出具有感染性的猪水泡病病毒(SVDV),并对其生物学功能进行了鉴定.该细胞系的建立为利用T7 RNA聚合酶转录系统体内高效拯救病毒提供了基础.该拯救病毒的策略使RNA拯救简化为一步快速的拯救方法,为进一步探索SVDV病毒致病的分子机制及研制新型SVD疫苗奠定了良好的基础.     

用基因芯片鉴别诊断四种水泡性疾病

为了对临床上症状非常相似的四种水泡性动物疾病进行大批量、快速准确的检测,我们尝试用基因芯片技术来对这四种水泡性疾病进行快速鉴别诊断.用Qiaquick 96 plate Kit对扩增的39个基因目的片段进行纯化,TE调配浓度至0.36μg/μL,于GMS417点样仪上按预选设计好的布阵方式点样,然后经紫外灯照射进行紫外交联等一系列处理.用Salmon进行非特异性杂交检测芯片点样的质量.大量抽提组织中FMDV和细胞上清液中的SVDV的总RNA,随机引物反转录的同时采用随机荧光标记法,标记FMDV和SVDV.标记好的cDNA超声波随机打断后进行特异性杂交,以最佳光扫描强度进行扫描,Imagene软件扫描结果,对两者信号进行对比,结合各病毒相对应的坐标,可鉴别诊断出四种动物的水泡性疾病病毒.本方法不但快速、灵敏、准确性高,而且可实现对大批量货物的集成化检测,满足我国快速通关的要求.     

用基因芯片鉴别诊断四种水泡性疾病

为了对临床上症状非常相似的四种水泡性动物疾病进行大批量、快速准确的检测,我们尝试用基因芯片技术来对这四种水泡性疾病进行快速鉴别诊断。用Qiaquick 96 plate Kit对扩增的39个基因目的片段进行纯化,TE调配浓度至0．36μg/μL,于GMS417点样仪上按预选设计好的布阵方式点样,然后经紫外灯照射进行紫外交联等一系列处理。用Salmon进行非特异性杂交检测芯片点样的质量。大量抽提组织中MDV和细胞上清液中的SVDV的总RNA,随机引物反转录的同时采用随机荧光标记法,标记FMDV和SVDV。标记好的cDNA超声波随机打断后进行特异性杂交,以最佳光扫描强度进行扫描,Imagene软件扫描结果,对两者信号进行对比,结合各病毒相对应的坐标,可鉴别诊断出四种动物的水泡性疾病病毒。本方法不但快速、灵敏、准确性高,而且可实现对大批量货物的集成化检测,满足我国快速通关的要求。     

小RNA病毒——新型的基因工程疫苗载体

小RNA病毒(Picornaviruses)是最小的RNA动物病毒,这类病毒的研究具有重要的意义,①小RNA病毒可以引起人和动物的许多严重疾病,如脊髓灰质炎、甲型肝炎、口蹄疫、水泡病等;②口蹄疫病毒(FMDV)是最早发现的动物病毒,脊髓灰质炎病毒(Poliovirus,PV)是最早被纯化并获得结晶的病毒。近来的研究发现,小RNA病毒尤其是PV有可能成为新的疫苗载体,可以插入某些已知或推测的病毒中和抗体决定簇的基因区域。本文仅就这方     

双重荧光RPA扩增方法快速鉴别两种血清型水泡性口炎病毒

本研究利用重组酶聚合酶扩增(RPA)技术,根据水泡性口炎病毒印第安纳型(VSV-IND)和新泽西型(VSVNJ)相对保守的L基因为靶序列设计并筛选出两套特异性的引物和探针,建立了快速鉴别检测水泡性口炎病毒两种血清型的双重荧光RT-RPA方法。试验结果表明,该方法特异性强,与猪水泡病病毒(SVDV)、口蹄疫病毒(FMDV)、蓝舌病病毒(BTV)、猪繁殖与呼吸综合征病毒(PRRSV)、牛病毒性腹泻病毒(BVDV)、猪瘟病毒(CSFV)等灭活抗原核酸无交叉反应;灵敏度高,最低可检测核酸浓度为12.7fg/μL;简便快速,反应时间短(15min),且重复性好。利用所建立方法对124份临床样品进行检测,结果与双重荧光RT-PCR一致。本文为VSV-IND和VSV-NJ的快速鉴别检测提供一种新方法,尤其适合现场检疫或基层实验室的快速检测。     

猪水泡病病毒VP1基因的克隆和表达

以猪水泡病病毒RNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了849bp的VP1基因,通过T-A克隆技术,将VP1基因片段克隆至pMD18-T克隆载体质粒中,构建SVDVVP1基因克隆重组质粒,进行核苷酸序列分析。然后亚克隆插入pBAD/ThioTOPO表达载体,经测序鉴定,筛选获得VP1基因正向插入、有正确读码框的阳性克隆,成功构建了猪水泡病病毒VP1基因重组表达载体。经L-Arabinose诱导表达,可稳定、高效地表达VP1蛋白抗原。SDS-PAGE结果表明,以终浓度为0.002%的L-阿拉伯醛糖进行诱导,5h后表达可达到高峰,表达蛋白为融合蛋白,质量约47.13kDa,表达产量约占菌体总蛋白的16%。Westernblotting检测表明,诱导的蛋白能与猪水泡病阳性血清发生特异性反应。融合蛋白中含有猪水泡病病毒VP1蛋白抗原,为应用该表达蛋白抗原制备SVD免疫血清学诊断试剂和新型疫苗构建奠定基础。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.