登革病毒2型E蛋白基因真核表达和DNA免疫的研究

将编码登革病毒2型(DV2)氨基末端80％的E蛋白的DNA片段克隆到真核表达载体pCXN2 AG强启动子下游,构建成DV2E重组真核表达质粒pCXN-E。间接免疫荧光显示其可在COS-7细胞中表达。ELISA法检测pCXN2-E DNA免疫BALB/c鼠血清中的E抗体变化和维持规律,结果显示三次免疫后2周已有抗体产生,15周时仍维持较高的水平;血清空斑减数中和实验显示其中和滴度高于1:640;流式细胞计数仪(FACS)检测DNA免疫鼠CD4~+、CD8~+T淋巴细胞变化情况,与注射空载体pCXN2的阴性鼠相比,CD4~+淋巴细胞水平略有上升。CD8~+细胞水平有较大升高(p<0.01);动物保护性实验结果显示,当用致死剂量登革病毒攻击免疫鼠时,其保护率为60％。以上结果表明:pCXN2-E在实验动物内表达出的DV2E蛋白可以诱导免疫动物的体液免疫和细胞免疫应答,尤其是MHC-I限制性杀伤性CD8~+T淋巴细胞水平的提高对清除病毒是十分有利的。因此,DV2 E DNA免疫为登革病毒DNA疫苗的发展进行了有益的探索。     

丙型肝炎病毒E2基因DNA 免疫实验动物研究

将编码丙型肝炎病毒(HCV)E2蛋白417～750位氨基酸的DNA片段克隆到真核表达载体pcDNA 3.1(-)中的CMVIE启动子下游,构建成HCV E2重组真核表达质粒pcE2.ELISA法检测pcE2 DNA免疫兔血清中的E2抗体变化和维持规律,结果显示免疫20d已有抗体产生,30d后开始进入高峰,40d时达到最高值,至第90d抗体水平保持平稳,抗体滴度达到11600左右.流式细胞计数仪(FACS)检测pcE2 DNA免疫鼠CD4+、CD8+T淋巴细胞变化情况,与注射空载体pCDNA3.1(-)的阴性鼠相比,CD4+淋巴细胞水平略有上升,CD8+细胞水平有较大升高,增幅达35.46%.免疫组化检测结果显示注射pcE2的小鼠组织中有明显的阳性着色,而注射pcDNA3.1(-)的对照组小鼠免疫组化结果为阴性.以上结果表明pcE2在实验动物内表达出的HCV E2蛋白可以引起免疫动物的体液免疫应答和细胞免疫应答,尤其是MHC-1限制性杀伤性CD8+T淋巴细胞水平的提高对清除病毒是十分有利的,因此HCV E2 DNA免疫有可能成为预防和治疗HCV感染的一条新途径.     

HCV E2蛋白诱导的体液免疫及CTL应答研究

应用PCR方法扩增出HCVE2基因编码417a.a～750a.a的DNA片段,克隆到原核表达载体pQE30 LacZ启动子下游,转化JM109菌株.在JM109菌株中诱导表达出N端含6个组氨酸的E2融合蛋白,用Ni-NTA-Superflow亲和层析柱纯化作为抗原免疫实验兔和BALB/c鼠.定期取免血,采用间接ELISA方法检测兔子体内针对E2的抗体水平和维持规律.结果显示,距初次免疫14d兔子体内已有抗体产生,直至免疫第55d抗体水平持续上升,之后抗体水平保持稳定,抗体滴度达到13200.六周后,取鼠脾脏制备淋巴细胞,定向刺激扩增后与经过重组真核表达质粒pCE2转染的P815细胞作用,利用LDH释放试验检测作用效果.在ET=2001的情况下,杀伤率超过30%.这些结果表明工程菌株表达的HCV E2蛋白具有良好的免疫原性,可以诱发免疫实验动物机体产生较高滴度的抗体及特异性CTL应答.由此我们认为E2蛋白是发展HCV预防工程蛋白疫苗的合适候选者.     

HCV E2蛋白诱导的体液免疫及CTL应答研究

应用PCR方法扩增出HCVE2基因编码417a．a-750a．a的DNA片段,克隆到原核表达载体pQE30 LacZ启动子下游,转化JM109菌株。在JM109菌株中诱导表达出N端含6个组氨酸的E2融合蛋白,用Ni-NTA-Superflow亲和层析柱纯化作为抗原免疫实验兔和BALB／c鼠。定期取兔血,采用间接ELISA方法检测兔子体内针对E2的抗体水平和维持规律。结果显示,距初次免疫14d兔子体内已有抗体产生,直至免疫第55d抗体水平持续上升,之后抗体水平保持稳定,抗体滴度达到1：3200。六周后,取鼠脾脏制各淋巴细胞,定向刺激扩增后与经过重组真核表达质粒pCE2转染的P815细胞作用,利用LDH释放试验检测作用效果。在E：T＝200：1的情况下,杀伤率超过30％。这些结果表明工程菌株表达的HCVE2蛋白具有良好的免疫原性,可以诱发免疫实验动物机体产生较高滴度的抗体及特异性CTL应答。由此我们认为E2蛋白是发展HCV预防工程蛋白疫苗的合适候选者。     

猪2型圆环病毒核酸疫苗的接种Balb/c小鼠实验免疫研究

为了筛选得到较理想的核酸疫苗,用pVAX1真核表达载体,以猪白介素18基因、猪2型圆环病毒内蒙株的衣壳蛋白和与复制相关蛋白基因为目的基因,构建了4个重组质粒,分别是pVAX1-ORF2、pVAX1-UL18ORF2、pVAX1-ORF2ORF1和pVAX1-IL18ORF2ORF1。用它们免疫6-8周龄Balb/c小鼠,进小鼠后一周首免,每隔19d免1次,共免3次;每10d采血1次,三免后10d杀鼠取脾。用ELISA方法检测小鼠血清抗体效价,流式细胞仪检测脾T淋巴细胞亚类CD4+、CD8+数量。统计学分析发现:4个核酸疫苗实验免疫组均是从最后一次采集的小鼠血清中,获得最高的抗体效价,与对照组PBS和空载体pVAX1相比都能产生一定的体液免疫反应,且以pVAx1-IL18ORF2ORF1核酸疫苗免疫小鼠产生的血清抗体效价最高;脾T淋巴细胞亚类数量测定发现,免疫实验组的CD4+与CD8+的T细胞亚类总数显著高于对照组(P<0．05),并以pVAX-1-IL18ORF2ORF1数值较高。结论是核酸疫苗pVAX1-IL18ORF2ORF1既能较好地刺激细胞免疫又能产生一定的体液免疫反应,相对较好,将作为候选核酸疫苗进行下一步本体动物免疫实验研究。     

HIV-2gag-gp105嵌合基因DNA疫苗对小鼠的免疫原性研究

利用真核表达载体pVAX1构建HIV-2 gag-gp105嵌合基因的重组质粒pVAX1gag-gp105,将其转入BHK21细胞中,利用间接免疫荧光方法检测其表达情况.进一步分别将核酸疫苗质粒pVAX1gag-gp105、对照组质粒pVAX1和PBS溶液经肌肉注射免疫BALB/c小鼠,检测免疫小鼠脾CD4+、CD8+T细胞亚群的数量,脾特异性CTL杀伤活性和血清抗体滴度.结果显示,重组核酸疫苗质粒pVAX1gag-gp105疫组小鼠脾CD4+、CD8+T细胞亚群的数值均比对照组高(P＜0.01),脾特异性CTL杀伤活性与对照组相比差异极显著(P＜0.01),血清抗体滴度显著高于对照组(P＜0.01).以上结果表明,HIV-2 gag-gp105嵌合基因DNA疫苗对BALB/c小鼠具有良好的体液和细胞免疫原性.     

HIV-1gag/IFNα-2b表达产物的生物活性研究

目的:将艾滋病病毒核心蛋白(gag)与干扰素(IFNα-2b)融合基因表达的融合蛋白作为免疫原免疫小鼠,动态观察小鼠的体液免疫、细胞免疫与CTL应答.方法:将IFNα-2b基因片段插入到gag基因的nt531位点,经脂质体转染与血凝素阴性蚀斑筛选,挑出重组痘苗病毒.经SDS-PAGE和Western blot鉴定表达产物.以小鼠为实验对象,用重组痘苗病毒vJ38gag/IFNα-2b免疫小鼠,用ELISA方法检测血清IgG抗体含量.用流式细胞仪测定小鼠外周血CD+4、CD+8T淋巴细胞计数.3H-TdR掺入法检测细胞毒性T淋巴细胞杀伤活性.结果:血清IgG抗体含量逐渐增高,实验组与对照组比较差异有显著性意义(p＜0.05).CD+4、CD+8T淋巴细胞计数、CTL检测实验组与对照组比较差异均有显著性意义(p＜0.05).结论:重组痘苗病毒vJ38gag/IFNα-2b能增强小鼠的体液免疫、细胞免疫和CTL应答.IFNα-2b可以作为免疫佐剂增强机体的免疫状态.     

冠状病毒HCoV-229E S1蛋白片段的免疫原性分析

目的表达和纯化HCoV-229E的S1蛋白片段（S1 417-547）,分析其诱导的免疫应答。方法将纯化蛋白免疫小鼠,ELISA检测小鼠血清特异性抗体以及血清IL-4和IFN-γ含量,流式细胞术检测小鼠脾脏T淋巴细胞亚群分布,观察其诱导的免疫应答。结果表达蛋白经金属螯合获得纯化,并诱导小鼠产生了高滴度的抗体。脾脏CD4^＋和CD8^＋比例均升高,CD4^＋/CD8^＋比值下降;免疫鼠血清IFN-γ和IL-4水平显著升高。结论成功构建了HCoV-229E S1蛋白的表达载体,并在BL21（DE3）中得到了高效表达,表达蛋白免疫小鼠后诱导了明显的细胞和体液免疫应答。     

IL-2与IL-7基因对犬细小病毒VP2 DNA疫苗在小鼠的免疫增强作用

[目的]探讨犬白细胞介素-2(cIL-2)与犬IL-7(cIL-7)基因对犬细小病毒(CPV) VP2蛋白DNA疫苗免疫增强的协同作用.[方法]利用含内部核蛋白体进入位点( IRES)的真核表达载体构建cIL-2和cIL-7双基因表达载体.然后利用本实验室构建的CPV VP2、cIL-2和cIL-7表达载体及本文构建的双基因表达载体,以不同组合对小鼠进行免疫,即VP2单免疫,VP2+cIL-2、VP2+cIL-7和VP2+cIL-2/cIL-7共免疫.通过ELISA方法检测免疫后不同时间小鼠血清VP2的抗体水平,并分析中和抗体的效价,通过细胞增殖实验检测免疫后小鼠脾脏淋巴细胞的增殖反应,并用ELISA方法测定小鼠淋巴细胞γ干扰素的表达水平.[结果]本实验构建的双基因表达载体结构正确,并能够介导cIL-2与cIL-7基因在真核细胞中进行同步分泌表达.小鼠免疫结果表明,VP2+cIL-2/cIL-7共免疫组小鼠血清的抗体滴度和中和抗体效价均显著高于VP2 +cIL-2和VP2+cIL-7共免疫组(P＜0.05).VP2+cIL-2/cIL-7共免疫组小鼠的淋巴细胞刺激指数比其它免疫组略有升高但尚未达到显著水平,然而γ干扰素的表达水平显著高于其它免疫组(P＜0.05).[结论]cIL-2和cIL-7基因对CPV VP2 DNA疫苗免疫原性的增强作用具有明显的协同效应.     

新疆栽培一枝蒿粗多糖对OVA抗体水平及T淋巴细胞亚群的影响

初步探讨新疆栽培一枝蒿未除蛋白的粗多糖(Un-deproteinization of cultivated Artemisia rupestris L.crude polysaccharides,UCARCP)对模式抗原卵清白蛋白(Ovalbumin,OVA)免疫小鼠后抗体水平以及T淋巴细胞亚群的影响。将UCARCP配伍OVA皮下免疫小鼠,初免1次,加强1次,铝佐剂为阳性对照组,间接ELISA法检测小鼠血清中IgG及亚类IgG_1、IgG_(2a)的抗体水平;流式细胞术检测脾细胞中CD3~+CD4~+和CD3~+CD8~+T淋巴细胞亚群的含量。结果显示,UCARCP能显著提高小鼠血清中Ig G、IgG_1、IgG_(2a)的抗体水平(P0.05),且与铝佐剂组相比无显著性差异(P0.05);UCARCP能显著促进CD3+CD4+和CD3+CD8+T细胞亚群的含量(P0.05),与铝佐剂组相比没有显著差异(P0.05)。新疆栽培一枝蒿未除蛋白的粗多糖能显著促进模式抗原OVA免疫后小鼠的体液免疫水平和细胞免疫水平,与铝佐剂相当。     

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.     

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.     

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.