阴道毛滴虫铁氧还蛋白基因的原核表达

目的在原核细胞中表达阴道毛滴虫铁氧还蛋白(ferredoxin,Fd)基因。方法构建阴道毛滴虫Fd基因的原核表达重组质粒pUC19-Fd,转化大肠埃希菌JM109感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白质表达。结果经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western blot)分析,重组质粒在大肠埃希菌中表达出Fd。结论在大肠埃希菌中表达出了Fd。     

肺炎链球菌SpxA蛋白的原核表达、纯化及多克隆抗体的制备

目的 构建肺炎链球菌SpxA蛋白的原核表达系统,制备其多克隆抗体.方法 设计引物,利用PCR技术扩增肺炎链球菌D39菌株的spxA基因,并插入表达载体pET-28a（＋）内,测序鉴定.重组质粒转化至大肠埃希菌BL21（DE3）中,以IPTG诱导表达含6个组氨酸标签的SpxA重组蛋白,经Ni-NTA亲和层析柱纯化后,以其为抗原免疫BALB/c小鼠制备多克隆抗体.用ELISA及Western印迹方法分别检测多克隆抗体的效价及特异性.结果 从大肠埃希菌中诱导出高表达的SpxA重组蛋白,纯化后免疫小鼠获得抗血清,ELISA测定其效价可达1：2 560 000以上,Western印迹结果显示其能特异性地作用于肺炎链球菌SpxA.结论 成功构建了pET-28a（＋）-spxA原核表达质粒,获得了高纯度的目的 蛋白和高滴度、高特异性的多克隆抗体.     

Nono蛋白的原核表达、纯化和多克隆抗体的制备

目的表达和纯化带多聚组氨酸（6×His）标签的Nono （ non-POU-domain-containing, octamer-binding protein ）融合蛋白并制备抗Nono多克隆抗体。方法构建pET-28a（＋）-Nono重组表达质粒,转入Rosetta（DE3）大肠埃希菌,以IPTG诱导6×His-Nono融合蛋白表达,经镍离子金属螯合树脂纯化后,用纯化出的蛋白免疫BALB／C小鼠制备多克隆抗体,并用ELISA检测多克隆抗体的效价,Western印迹检测多克隆抗体的特异性。结果在大肠埃希菌中诱导出高水平表达的His-Nono融合蛋白,经亲和树脂纯化后免疫小鼠,获得了高特异性的抗Nono抗血清。结论成功构建pET-28a（＋）-Nono原核表达质粒,表达并纯化出高纯度的目标蛋白,制备出高滴度、高特异性的多克隆抗体。     

阴道毛滴虫黏附蛋白65-3基因原核表达质粒的构建及表达

质粒 pMD-18T-ap65-3 经 Xho I 和 BamH I 双酶切, 1.0% 凝胶电泳后纯化回收阴道毛滴虫黏附蛋白 65-3 基因,定向克隆至原核表达载体 pET-32a( ), 构建原核表达质粒 pET32a-ap65-3. 重组质粒转化大肠埃希菌 JM109 感受态细胞中, 筛选阳性克隆, 进行PCR﹑酶切及测序鉴定正确后, 将重组质粒 pET32a-ap65-3 转化于大肠埃希菌 BL21 中,异丙基-β-D硫代半乳糖苷 (IPTG) 诱导重组蛋白表达后利用十二烷基磺酸钠一聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和免疫印迹 (Western blot) 对重组蛋白进行分析鉴定. 本实验为研究阴道毛滴虫病的致病机制及蛋白生物学功能奠定了基础.     

小鼠膜型抗衰老蛋白Klotho基因特异片段的克隆、原核表达及多克隆抗体制备

目的克隆小鼠膜型抗衰老蛋白Klotho基因特异片段,制备小鼠Klotho多克隆抗体。方法以小鼠基因组为模板进行PCR,克隆了小鼠膜型抗衰老蛋白Klotho基因外显子Ⅳ部分序列,经BamH I和Nhe I双酶切后定向克隆到质粒pET-GST中,构建原核表达质粒pET-GST-Klotho,转化大肠埃希菌BL21（DE3）,用IPTG诱导表达。以重组GST-Klotho融合蛋白免疫家兔,制备Klotho多克隆抗体。结果表达产物经SDS-PAGE检测表明,在大肠埃希菌中成功表达了GST-Klotho融合蛋白,GST-Klotho融合蛋白表达量占菌体总蛋白的15％左右;另外通过ELISA法测得抗血清抗体效价约为1：10000,Western印迹分析验证了抗体特异性。结论GST-Klotho融合蛋白的表达和Klotho多克隆抗体的制备为进一步研究Klotho蛋白在小鼠体内的表达模式以及相关抗衰老药物的研制奠定了基础。     

CTX-M-3型超广谱β-内酰胺酶的表达、纯化及多克隆抗体的制备

目的 将肺炎克雷伯菌所产CTX-M-3型超广谱β-内酰胺酶(extended-spectrum β-lactamase,ESBLs)进行表达、纯化及其多克隆抗体的制备.方法 将保存的重组质粒pET-28a(+)/CTX-M-3在大肠埃希菌BL21( DE3)中原核表达.IPTG诱导CTX-M-3融合蛋白表达,利用镍琼脂糖凝胶亲和柱层析纯化蛋白,用纯化的CTX-M-3型ESBLs酶蛋白免疫小鼠制备多克隆抗体.结果 可溶性检测表明表达产物以可溶性形式(上清中)和包涵体形式(沉淀中)两种形式存在.通过蛋白表达条件的优化实验,SDS-PAGE电泳结果显示18℃,0.8 mmol/L IPTG诱导24 h的CTX-M-3重组蛋白的可溶性表达最佳.SDS-PAGE电泳和Western-blot检测表明获得纯化的重组32 kD蛋白.免疫获得的CTX-M-3型ESBLs酶蛋白抗血清的效价为1∶32.结论 pET-28a(+)/CTX-M-3表达载体在大肠埃希菌BL21( DE3)中表达,用His亲和层析柱纯化可获得高纯度可溶性的重组蛋白,成功制备了效价较高的抗CTX-M-3型ESBLs酶蛋白多克隆抗体.     

抗博尔纳病病毒核蛋白多克隆抗体的制备和鉴定

目的利用重组博尔纳病病毒核蛋白进行动物免疫,制备多克隆抗体并对其进行鉴定。方法将重组载体pET14b-p40转化至感受态大肠埃希菌I,PTG诱导融合蛋白的表达,His-tag亲和层析纯化重组核蛋白并作为抗原免疫新西兰大白兔,收集免疫后血清,制备和纯化多克隆抗体,ELISA测定抗体效价,并进行Western-blot鉴定。结果成功制备出核蛋白多克隆抗体,ELISA检测效价高达1︰256000;该抗体与原核和真核系统中表达的核蛋白均能发生特异性反应。结论成功制备了效价和特异性良好的抗重组核蛋白多克隆抗体,为博尔纳病病毒血清免疫学检测方法的建立奠定了基础。     

金黄色葡萄球菌LDH多克隆抗体制备及其交叉反应性

目的:在原核载体中表达、纯化金黄色葡萄球菌乳酸脱氢酶,免疫小鼠获得多克隆抗体,使用多克隆抗体分析与其它菌种的交叉反应性。方法:复苏p ET28a-ldh/Bl21重组菌,IPTG诱导重组融合蛋白表达、以抗HIS标签的单克隆抗体进行western-blot鉴定重组蛋白。使用纯化的重组蛋白以及相应的佐剂免疫小鼠,利用ELISA测定血清抗体效价,并使用小鼠抗血清进行免疫印迹法鉴定重组蛋白的反应原性及其与金黄色葡萄球菌、表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌的交叉反应性。结果:SDS-PAGE电泳在39 KDa处可见目的条带,免疫印迹法验证了重组LDH的表达,纯化后获得2.8 mg重组蛋白。纯化蛋白免疫小鼠能诱导产生特异性体液免疫应答,ELISA检测特异性Ig G效价为1:50000,western-blot鉴定显示所制备的多克隆抗血清能分别识别金黄色葡萄球菌重组及天然乳酸脱氢酶,但不识别表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌中天然乳酸脱氢酶。结论:纯化的LDH具有良好的免疫活性,免疫小鼠获得高滴度的多克隆抗体。使用多克隆抗体western-blot显示与其它菌种LDH不存在交叉反应性,为后续使用该重组蛋白进行金黄色葡萄球菌感染的诊断研究奠定基础。     

诺如病毒NV衣壳蛋白VP1表达纯化及多克隆抗体的制备

目的获得纯化的诺如病毒(NV)衣壳蛋白VP1,免疫动物制备多克隆抗体。方法提取粪便样品中诺如病毒RNA,逆转录得到cDNA文库,通过PCR扩增获取VP1基因序列,构建到大肠埃希菌原核表达系统中诱导表达重组VP1蛋白。使用镍柱亲和层析法对重组蛋白进行纯化,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和考马斯亮蓝法(BSA)对重组蛋白的纯度与浓度进行分析,以重组的VP1蛋白为抗原,免疫雄性SPF级SD大鼠获得多抗血清,用ELISA测定抗体效价、Western blot检测抗体特异性。结果成功地构建出重组表达载体VP1-pET28a,并将其在大肠埃希菌BL21(DE3)中稳定地表达诱导重组蛋白。ELISA测其多抗血清的平均效价为1∶200 000,Western blot检测抗体在原核和真核特异性很高。结论本实验成功地利用原核表达系统表达诺如病毒衣壳蛋白VP1,为进一步研究诺如病毒的诊断和疫苗开发提供了条件。     

中国旱獭β2m基因的克隆、原核表达及多克隆抗体的制备

目的克隆、原核表达中国旱獭β2m基因,并制备多克隆抗体。方法利用RT—PCR技术从中国旱獭脾细胞中扩增β2m基因,克隆至pET28a（＋）质粒,构建原核表达载体pET-28a（＋）-CWβ2m,再转化宿主菌Rosetta（DE3）pLacI诱导其表达。使用切胶回收纯化目的蛋白,将纯化蛋白免疫家兔制备多克隆抗体,并采用酶联免疫吸附实验检测抗体的灵敏度和特异性。结果克隆出的中国旱獭β2m基因,与GenBank已公布的土拨鼠的碱基序列一致;Western印迹结果显示克隆的β2m基因能在大肠埃希菌中高效表达,免疫家兔获得了高效价的多克隆抗体。结论成功克隆了中国旱獭β2m基因,在原核宿主中进行了高效表达,获得的多克隆抗体具有较高的效价。为人工制备MHC-Ⅰ类分子复合物,深入研究嗜肝病毒感染过程中特异性CTL应答和效应机制奠定了基础。     

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.     

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.     

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.     

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。     

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.     

（鱼句）亚科花（鱼骨）型鱼类骨骼系统的比较

对我国花型Hemibarbuspattern鱼类作了骨骼系统比较,结果表明,此类型鱼类脑颅较长,副蝶骨平直或稍弯曲,眶蝶骨腹纵嵴发达(铜鱼Coreius septentrionalis例外),下颞窝和咽突中等大,基枕骨后突发达;脑颅中的上筛骨的后突、侧筛骨的外筛突,蝶耳骨的外突、上耳骨的后突、围眶骨和后颞窝等均有明显的差异;咽颅中的舌颌骨、尾舌骨、鳃盖骨和下咽齿的列数等又有显著的区别;附肢骨骼中的腰带骨、脊椎骨中的复合神经骨和第4椎骨腹侧的悬器等也有不同之处。据此,这些差异和区别可作为属间或种间的分类依据。