嗜肺军团菌mip/ctxB融合基因体外表达与动物免疫试验的免疫原性研究

ＰＣＲ扩增嗜肺军团菌mip基因和霍乱弧菌ctxB基因,克隆入载体pcDNA3.1(+),重组子经限制性酶切分析、PCR、序列分析鉴定正确后,命名为pcDNA3.1-mip/ctxB.脂质体法将重组质粒pcDNA3.1-mip、pcDNA3.1-mip/ctxB转染NIH3T3细胞,用免疫荧光法和蛋白质印迹鉴定瞬时表达和稳定表达产物,结果发现：重组质粒成功转入细胞并获得短暂表达,稳定转染细胞分别在24 ku和35 ku处检测到阳性杂交信号.将pcDNA3.1-mip、pcDNA3.1-mip/ctxB作为DNA疫苗免疫BALB/c小鼠,检测免疫小鼠体内抗原特异性抗体水平、脾淋巴细胞增殖活性、IFN-γ产生水平、细胞毒性T淋巴细胞(CTL)杀伤活性等体液免疫和细胞免疫反应的指标,评价疫苗的免疫原性.结果发现：各实验组均检测到免疫原性,pcDNA3.1-mip/ctxB免疫组的免疫原性高于pcDNA3.1-mip免疫组,有显著性差异(P＜0.01).研究结果为mip/ctxB融合基因DNA疫苗的研制提供了初步的实验依据.     

汉坦病毒核蛋白基因疫苗的初步研究

探讨了汉坦病毒核蛋白(NP)S基因疫苗的免疫效果。构建重组真核表达质粒pcDNA3.1 S,直接肌注免疫BALB/c小鼠,分别用ELISA法检测血清特异性抗体的变化,MTT法检测T细胞增殖反应,以及用ELISAkit检测免疫鼠脾细胞上清液中细胞因子IL-4和IFN-γ的动态变化,从而了解免疫鼠的体液免疫反应和细胞免疫反应。pcDNA3.1 S接种组的小鼠血清抗体水平、T细胞增殖反应以及细胞因子IL-4和IFN-γ的产生水平均较对照组pcDNA3.1 空载体接种组的要高。编码核蛋白的S基因的核酸免疫可同时启动机体的细胞免疫和体液免疫反应,S基因是汉坦病毒核酸疫苗的一个比较理想的侯选抗原基因。     

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.     

人乳头瘤病毒l6型E6-E7融合蛋白真核表达载体的构建与鉴定

目的构建人乳头瘤病毒l6型(HPV16)E6-E7融合蛋白真核表达载体,为研究其基因疫苗免疫活性奠定实验基础。方法 PCR扩增HPV16 E6-E7基因片段,将其连接到真核表达载体pcDNA3.1(+),构建真核表达载体pcDNA3.1(+)/HPV16 E6-E7,双酶切及测序鉴定。将质粒转染HeLa细胞,RT-PCR鉴定E6-E7基因在HeLa细胞中的表达。提取质粒免疫小鼠,利用免疫组化方法检测在其肌肉组织中的表达。结果成功构建了真核表达载体pcDNA3.1(+)/HPV16 E6-E7;在转染pcDNA3.1(+)/HPV16 E6-E7的细胞中检测到HPV16 E6-E7基因。在免疫该质粒的小鼠肌肉组织中可以检测到该质粒的蛋白表达。结论成功的构建的了真核表达载体pcDNA3.1(+)/HPV16 E6-E7,该载体能在HeLa细胞内以及小鼠骨骼肌细胞内有效表达。     

增加CpG基元的汉坦病毒核蛋白基因疫苗的免疫作用

以往的研究表明非甲基化胞嘧啶鸟嘌呤二核苷酸为基元构成的特定结构是一种很有希望的候选疫苗佐剂.为了研究增加CpG基元的汉坦病毒核蛋白基因疫苗的免疫作用,在本实验中,将重组真核表达质粒pcDNA3.1+S(ISS)直接肌注免疫BALB/c小鼠,分别用ELISA法检测血清特异性抗体的变化,MTT法检测T细胞增殖反应,以及用ELISA kt检测免疫鼠脾细胞上清液中细胞因子IL-4和IFN-γ的动态变化,从而了解免疫鼠的体液免疫反应和细胞免疫反应.结果显示pcDNA3.1+S(ISS)接种组的小鼠血清抗体水平、T细胞增殖反应以及细胞因子IFN-γ的产生水平较对照组pcDNA3.1+S和空载体pcDNA3.1+接种组的要高.而细胞因子IL-4较对照组却无明显变化.由此可见,增加CpG基元的质粒能够增强其所诱导的免疫反应,可用于那些需要借助强有力细胞免疫来清除病原体的疫苗研制中.     

羊痘病毒P32基因真核表达载体的构建、表达及其免疫原性

通过PCR方法扩增全长P32基因和截去跨膜区的P32基因(MP32),将其分别克隆到真核表达载体pcDNA3.1( )和已插入CpG序列的pcDNA3.1-CpG中,构建pcDNA3.1-P32、pcDNA3.1-CpG-P32和pcDNA3.1-CpG-MP32质粒;用脂质体法转染BHK-21细胞,通过间接免疫荧光(IFA)试验验证其表达效果;经肌肉免疫注射健康BALB/c小鼠,用间接ELISA法检测抗体;在免疫后的第3、5周取免疫小鼠的脾细胞,用流式细胞仪检测CD4 和CD8 T细胞亚群.结果所构建的真核表达载体在BHK-21细胞中都能表达P32蛋白;免疫小鼠血清在免疫第2周后均能检测到羊痘特异性IgG抗体;免疫组小鼠脾脏CD4 T细胞数目和CD4 /CD8 T细胞比值明显高于对照组.结果提示,所构建的真核载体可诱导小鼠产生特异性体液免疫应答,并能刺激小鼠产生较强的细胞免疫应答.     

幽门螺杆菌Lpp20-IL2核酸疫苗免疫活性

【目的】观察pcDNA3.1(+)/Lpp20-IL2免疫C57BL/6小鼠后所产生的体液免疫和细胞免疫应答水平,为研制高效、新型的幽门螺杆菌核酸疫苗提供实验依据。【方法】构建pcDNA3.1(+)/Lpp20-IL2重组载体,并转染HeLa细胞,用Western-blot观察鉴定其在真核细胞得到表达后免疫C57BL/6小鼠,ELISA间接法测定小鼠血清中抗Lpp20IgG抗体水平,ELISA双抗体夹心法检测脾淋巴细胞培养上清中IFN-γ、IL4水平,MTT比色法检测脾淋巴细胞增殖反应,免疫荧光组化法检测Lpp20蛋白在小鼠肌肉组织中的表达情况。【结果】成功构建了pcDNA3.1(+)/Lpp20-IL2真核表达载体,且重组质粒能在HeLa细胞内有效表达目的蛋白;小鼠接种pcDNA3.1(+)/Lpp20-IL2核酸疫苗后能产生特异性IgG抗体,8w后ELISA测定血清抗体A450值明显升高。核酸疫苗pcDNA3.1(+)/Lpp20-IL2免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ、IL4含量明显升高。pcDNA3.1(+)/Lpp20-IL2和pcDNA3.1(+)/Lpp20核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数明显高于空质粒组和PBS组。Lpp20蛋白在小鼠肌肉组织中能够有效表达。【结论】幽门螺杆菌Lpp20-IL2融合基因核酸疫苗和Lpp20单基因核酸疫苗均能刺激机体产生较强细胞免疫应答和体液免疫应答,且前者能诱导更强的细胞免疫应答。     

HIV-2 gag-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。     

小鼠EVL 基因的克隆和真核表达载体的构建

目的:构建小鼠EVL(Ena/VASP like)基因的真核表达载体,为深入研究EVL的功能奠定基础.方法:采用PCR方法,从小鼠cDNA文库中,扩增出1245bp的EVL编码区片段,经电泳、胶回收后连接入pMD- 18T载体中,测序鉴定正确.用BamHI和HincⅡ双酶切,定向克隆EVL编码区片段到真核表达载体pcDNA3.1中,用限制性内切酶酶切鉴定重组质粒正确后.将重组质粒转染入HELA细胞中,以RT-PCR检测EVL的mRNA的表达,以Western Blot检测EVL蛋白的表达.结果:酶切鉴定结果显示小鼠EVL编码区基因被成功克隆入真核表达载体pcDNA3.1中;RT-PCR和Western Blot结果以及免疫荧光染色显示Hela细胞中有EVL的mRNA和蛋白的表达.结论:成功获得pcDNA3.1 -EVL的真核表达载体,为进一步深入研究EVL蛋白的功能奠定了基础.     

小鼠白细胞介素33真核表达质粒的构建及表达

克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应（RT-PCR）扩增小鼠IL-33基因,酶切后插入pcDNA^TM3.1/myc HisA构建其真核表达质粒pcDNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法（western blotting）检测目的基因表达。结果显示,pcDNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33cDNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因cDNA,并构建其真核表达质粒。     

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.