H5N1亚型禽流感病毒神经氨酸酶基因的克隆与表达

根据已知H5N1亚型禽流感病毒神经氨酸酶基因(na)序列设计、合成克隆引物。自H5N1亚型病毒感染的鸡胚尿囊液中提取总RNA,反转录后采用高可信度DNA聚合酶(PyobestTMDNAPolymerase)扩增na基因,采用Invitrogen定向表达系统(ChampionTMpETdirectionalTOPOexpressionsystem)进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组神经氨酸酶,分子量约53.8kDa。分析重组NA的免疫反应性和免疫原性,结果表明:重组NA能与H5N1亚型病毒抗血清发生特异性结合,且其免异动物后能诱导机体产生特异性抗体,具有良好的抗原性。     

禽流感病毒H5N1血凝素基因和神经氨酸酶基因在大肠杆菌中的表达

目的：克隆、表达和鉴定禽流感病毒H5N1血凝素基因（hemagglutinin,HA）和神经氨酸酶基因（neuramidinase,NA）序列,为制备抗体和基因工程疫苗打下基础。方法：在成功克隆禽流感病毒H5N1全长HA、NA基因并测序的基础上,将部分基因序列克隆到表达载体pET32a（＋）上,全基因序列克隆到表达载体pGEX4T-1上,构建了重组表达质粒pET32a（＋）/HA(49~1587bp)、pET32a（＋）/NA(121~1141bp)、pGEX4T-1/HA、pGEX4T-1/NA,转化大肠杆菌BL21/rosetta,IPTG诱导表达,利用Ni2＋亲和层析柱和GSTra P4B亲和层析柱对重组蛋白进行纯化,并用Western blotting和ELISA方法检测其抗原性。结果：重组蛋白在大肠杆菌中可以高效达,SDS PAGE显示其相对分子质量与预计大小一致,蛋白纯度占总蛋白的90％以上。ELISA和Western blotting实验证实,重组蛋白具有良好的抗原性。结论:成功克隆和表达了禽流感病毒H5N1 HA、NA基因序列,为禽流感病毒H5N1诊断试剂和疫苗的开发等进一步的研究奠定了基础。     

禽流感病毒H9N2血凝素基因和神经氨酸酶基因在真核酵母菌中的表达

目的:克隆、表达和鉴定禽流感病毒H9N2血凝素基因(hemagglutinin,HA)和神经氨酸酶基因(neuramidinase,NA)序列,为制备抗体和基因工程疫苗打下基础。方法:在成功克隆禽流感病毒H9N2全长HA、NA基因并测序的基础上,将部分基因序列克隆到表达载体pMETA上,构建了重组表达质粒pMETA/HA(52～1545bp),pMETA/NA(121～1254bp),电转化真核酵母菌pMAD16,甲醇诱导表达,利用Ni2+亲和层析柱对重组蛋白进行纯化,并用Western blotting和ELISA方法检测其抗原性。结果:重组蛋白在酵母菌中可以高效表达,SDS-PAGE显示蛋白表达后形成了二聚体,蛋白纯度占总蛋白的95%以上,ELISA和Western blotting实验证实,重组蛋白具有良好的抗原性。结论:成功克隆和表达了禽流感病毒H9N2HA、NA基因序列,为禽流感病毒H9N2诊断试剂和疫苗的开发等进一步的研究奠定了基础。     

流感病毒神经氨酸酶的表达及其在药物筛选中的应用

A型流感病毒H5N1神经氨酸酶奥司他韦敏感型及耐药突变型基因经优化后,克隆于pcDNA4/TO 表达载体,并转染T-REx293 建立稳定细胞株,经四环素诱导能特异表达神经氨酸酶,其活性被特异性抑制剂奥司他韦所抑制。利用该稳定细胞株制备的神经氨酸酶,对3000多种天然产物和中药提取物进行了筛选,结果显示黄芩甙和黄芩素对奥司他韦敏感型神经氨酸酶和耐药型神经氨酸酶具有相似的抑制作用。该神经氨酸酶制备方法安全、简便、稳定,有利于建立神经氨酸酶抑制剂的高通量筛选方法。A型流感病毒H5N1神经氨酸酶奥司他韦敏感型及耐药突变型基因经优化后,克隆于pcDNA4/TO 表达载体,并转染T-REx293 建立稳定细胞株,经四环素诱导能特异表达神经氨酸酶,其活性被特异性抑制剂奥司他韦所抑制。利用该稳定细胞株制备的神经氨酸酶,对3000多种天然产物和中药提取物进行了筛选,结果显示黄芩甙和黄芩素对奥司他韦敏感型神经氨酸酶和耐药型神经氨酸酶具有相似的抑制作用。该神经氨酸酶制备方法安全、简便、稳定,有利于建立神经氨酸酶抑制剂的高通量筛选方法。     

高致病性A(H5N8)亚型禽流感病毒NA基因核酸检测方法的建立

建立以Real-time PCR为基础的新型高致病性A(H5N8)亚型禽流感病毒NA基因检测方法。针对2016年6月起频繁暴发的H5N8禽流感疫情,从GenBank和Global Initiative on Sharing All Influenza Data(GISAID)下载2014年以来的H5N8亚型禽流感病毒的NA序列,通过序列比对,在相对保守区域设计适用于实时荧光逆转录聚合酶链式反应(rRT-PCR)的引物和探针。选用28株不同NA亚型的流感病毒进行特异性验证,结果显示本文设计的引物探针组合能够特异性检测高致病性H5N8亚型禽流感病毒的NA基因。灵敏度检测结果显示,本文设计的引物探针组合能检出最低23个拷贝的RNA。本文建立了高致病性H5N8亚型禽流感病毒NA基因特异性荧光定量检测方法,与世界卫生组织(WHO)推荐的A型流感病毒M基因、H5基因检测引物探针的最低检测限一致,可以组合用于H5N8亚型禽流感病毒的检测。     

流感病毒疫苗(含甲型H1N1)生产制备和关键技术

1 流感与流感病毒 流行性感冒(以下简称"流感")由甲、乙、丙3种流感病毒引起.流感病毒易变异,传播迅速,其中甲型流感病毒最易引发大规模流行.根据甲型流感病毒表面抗原血凝素(HA)和神经氨酸酶(NA)结构及其基因特性的不同分成许多亚型,至今已发现的血凝素有16个亚型(H1～H16).神经氨酸酶9个亚型(N1～N9).     

神经氨酸酶茎部氨基酸缺失对H5N1亚型禽流感病毒生物学特性的影响

近年来H5N1亚型禽流感病毒(AIV)神经氨酸酶(NA)茎部15~20个氨基酸的自发缺失时有报道,突变对于AIV生物学特性的影响还没有得到系统研究。应用反向遗传操作技术,拯救获得5株具有不同NA茎部长度的H5N1/PR8重组AIV。重组病毒的内部基因和血凝素(HA)基因来源相同,NA基因来源不同,并在NA茎部进行20个氨基酸的删除或添加突变。通过研究其生物学特性发现,5株重组病毒在SPF鸡胚中繁殖良好,其EID50、MDT和平均病毒滴度相似;NA茎部长短影响病毒的解凝能力,长茎病毒红细胞解脱能力比短茎病毒强;NA茎部15或20个氨基酸删除突变提高了重组病毒在MDCK细胞上的繁殖能力,短茎病毒释放出的病毒粒子数量是长茎病毒的10~100倍,释放时间提前6~10h,短茎病毒在MDCK细胞上形成的空斑也明显比长茎病毒的空斑大。实验结果揭示了AIV NA茎部氨基酸缺失突变的生物学意义,NA茎部15或20个氨基酸删除突变增强了AIV的细胞适应性,可能与现阶段H5N1亚型AIV宿主范围进一步扩大有关。利用反向遗传技术成功拯救了5株H5N1/PR8重组流感病毒,为流感病毒基因功能研究和重组疫苗研究建立了技术平台。通过对AIV NA茎部氨基酸的删除突变提高了病毒在MDCK细胞上的繁殖产量,为流感病毒细胞苗的生产提供了新的思路。     

新型H7N9亚型禽流感病毒多重荧光RT-PCR快速检测方法的建立

为研制新型H7N9亚型禽流感病毒快速检测试剂盒,根据GenBank中公布的新型H7N9亚型(2013年)禽流感病毒血凝素抗原(HA)和神经氨酸酶抗原(NA)的基因序列,设计两套特异性的引物和探针,建立基于TaqMan探针的多重荧光RT-PCR快速检测新型H7N9亚型禽流感病毒方法。实验结果表明,该方法灵敏度高、特异性好,能够检测到最低浓度为10拷贝/μL数量级的阳性标准品,不但能够将禽流感病毒H7亚型与H1、H3、H5、H6和H9亚型区分开,而且可将新型N9亚型禽流感病毒与原有的N9亚型区分开。采用该方法对珠海市2 700份样品的检测结果与预期结果一致,证实该方法具有较好的推广应用前景。     

禽流感病毒M1蛋白的表达及其免疫反应性分析

根据已发表的禽流感病毒M1基因序列设计合成PCR克隆引物,自接种H5N1亚型病毒的鸡胚组织中提取RNA,反转录后采用高可信度DNA聚合酶(PyobestTM DNA Polymerase)经PCR扩增M1基因,采用Invitrogen定向表达系统(ChampionTM pET directional TOPO expression system)进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组蛋白,分子量约29.8 kDa.采用单克隆抗体和阳性血清经ELISA、阻断ELISA、免疫印迹分析重组蛋白的免疫反应性.结果发现重组M1蛋白能与单克隆抗体和阳性血清发生特异性结合,且此结合能被天然病毒抗原阻断.研究表明重组蛋白M1具有良好免疫反应性.     

利用基因芯片技术区分禽流感病毒主要亚型

[目的]研制可同时区分AIV的H5、H7、H9血凝素亚型及N1、N2神经氨酸酶亚型的基因诊断芯片.[方法]分别克隆了禽流感病毒的M基因,H5、H7、H9亚型HA基因,N1、N2亚型NA基因以及看家基因GAPDH的重组质粒.以重组质粒为模板,用PCR方法扩增制备探针,纯化后点于氨基修饰的片基上,制备基因芯片.在PCR过程中对待检样品进行标记,然后与芯片杂交,洗涤,扫描并进行结果分析.[结果]结果显示检测探针可特异性的与相应的标记样品进行杂交,呈现较强的杂交信号,且无交叉杂交.同时用RT-PCR、鸡胚接种和基因芯片方法对H1-H15亚型AIV参考毒株、30份人工感染样品、21份现地疑似样品进行检测,结果发现,对人工感染样品芯片检测方法与鸡胚接种和RT-PCR的符合率分别为100%和96%,现地样品符合率为100%.[结论]研究表明该方法可用于同步鉴别部分主要流行的禽流感亚型,是一种有效的新方法.     

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.     

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.     

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.     

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。     

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.     

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.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.