乙型肝炎病毒全长PreS蛋白基因在酵母中的表达

应用直接提取法从武汉地区乙型肝炎病人患者阳性血清中提取HBV基因组,以此作为模板,用引物PCR方法获得全长preS基因,该片段的DNA大小1 203bp.克隆到pUCm-T载体中,应用M13通用引物进行序列测定,与中国HBV标准株序列比较,证实基因型为Adr亚型.有24个核苷酸不同,preS基因包含3个起始密码ATG分别为preS1,preS2,和S的翻译起点.然后将preS基因克隆到毕赤酵母表达载体pPIC9K中,将Sal I线性化的pPIC9K-preS质粒用电击法导入巴斯德-毕赤酵母GS115His-中.通过MD-G418平板筛选和5'AOX1和3'AOX1引物PCR鉴定获得稳定重组HBV全长preS基因的His+Mut+酵母工程菌株.此酵母菌株在合适的培养条件和甲醇诱导下高效表达产生全长PreS蛋白并可分泌到培养液中,SDS-PAGE显示培养液中含有分子量约为48kDa的带;微孔ELISIA法证实表达并分泌到培养液中的PreS蛋白能够与HBV抗体阳性血清发生特异性反应.     

一种新型巴斯德毕赤酵母表达载体的构建及甘露聚糖酶的表达

目的:构建以带自身启动子的蔗糖转化酶基因(suc2)为选择标记的载体,用于外源基因在巴斯德毕赤酵母中的正确分泌表达。方法:根据已发表的蔗糖转化酶基因序列设计并合成1对引物,应用PCR技术,以啤酒酵母INVSC1总DNA为模板,扩增出包含自身启动子和终止区序列的suc2基因。将该基因与毕赤酵母表达载体pPIC9K连接,构建了以suc2为选择标记的表达载体pPIC12K。将甘露聚糖酶基因man克隆入载体pPIC12K,用PEG/LiCl法转化毕赤酵母GS115菌株。以蔗糖为惟一碳源筛选转化子,利用底物平板检测筛选到的转化子中man基因的表达,并对重组表达菌株进行连续传代实验。结果:部分转化子周围产生明显的水解圈,证明甘露聚糖酶已经得到分泌表达;对重组表达菌株的连续传代实验证实了该表达载体具有良好的遗传稳定性。结论:以带自身启动子的suc2基因为选择标记的表达载体构建成功,并且这个新型表达载体能够对外源基因进行稳定有效的分泌表达。     

苦瓜MAP30基因的毕赤酵母表达载体构建及重组菌株的筛选

目的:从苦瓜中克隆MAP30全长基因,并将该基因连接至表达载体pPIC9中,建立酵母菌落PCR筛选方法。方法采用改良SDS法从苦瓜表皮中提取基因组DNA,设计特异性的引物,通过PCR技术扩增出全长861bp的MAP30基因。该基因经XhoⅠ和EcoRⅠ双酶切,连接至毕赤酵母表达载体pPIC9中。重组载体转化GS115菌株,运用菌落PCR鉴定重组菌株。结果:基因测序表明,该基因已成功插入酵母表达载体pPIC9α-factor分泌信号下游,同源性分析表明该基因与GeneBank(AF284811)的核苷酸同源性达99.9%,氨基酸同源性达100%。菌落PCR显示外源基因已整合入酵母GS115菌株中。结论:成功地克隆了MAP30全长基因,并构建了含MAP30基因的重组毕赤酵母表达载体,并获得了整合菌株,为下一步研究奠定了基础。     

米曲霉木聚糖酶基因的克隆及其在毕赤酵母中的表达

目的:构建米曲霉木聚糖酶基因的真核表达载体,并转化巴斯德毕赤酵母,进行分泌表达。方法:以米曲霉总RNA为模板,根据已知的米曲霉木聚糖酶基因序列设计引物,采用RT-PCR技术克隆木聚糖酶基因cDNA序列,将其与pPIC9K质粒连接构建表达载体后转化毕赤酵母,经MM/MD快慢斑筛选,得到Muts型重组子,进行甲醇诱导表达。结果:克隆得到的cDNA序列全长666 bp,连续编码221个氨基酸;阳性克隆子在诱导培养数天后,将菌液点于RBB-木聚糖平板上,产生了明显的透明圈,表明重组木聚糖酶在毕赤酵母中获得表达。结论:木聚糖酶基因的真核表达载体构建成功,并能够在毕赤酵母中表达。     

细极链格孢菌peaT2基因在毕赤酵母中的表达及蛋白功能确定

从细极链格孢菌表达文库获得阳性克隆子,序列分析表明,克隆的DNA片段中含有完整的开放阅读框架,将该基因命名为peaT2(GenBank登录号为EF212880)。用PCR法扩增peaT2基因的编码序列并亚克隆到毕赤酵母表达系统的表达载体pPIC9K上,得到重组质粒pPIC9K/peaT2。重组质粒经SacⅠ线性化后用电穿孔法导入到毕赤酵母(Pichia pastoris)GS115中,采用MD、G418-YPD平板和PCR法筛选Mut+表型,获得了分泌表达的重组毕赤酵母。随机挑取一菌株作为表达菌,用甲醇诱导PeaT2蛋白表达。SDS-PAGE及Western blot检测结果均表明PeaT2在毕赤酵母中成功地分泌表达。用peaT2基因的表达蛋白处理小麦种子,生物测定表明,表达蛋白能明显促进小麦的生长,具有蛋白激发子作用。     

细极链格孢菌peaT2基因在毕赤酵母中的表达及蛋白功能确定

从细极链格孢菌表达文库获得阳性克隆子,序列分析表明,克隆的DNA片段中含有完整的开放阅读框架,将该基因命名为peaT2(GenBank登录号为EF212880)。用PCR法扩增peaT2基因的编码序列并亚克隆到毕赤酵母表达系统的表达载体pPIC9K上,得到重组质粒pPIC9K/peaT2。重组质粒经SacⅠ线性化后用电穿孔法导入到毕赤酵母(Pichia pastoris)GS115中,采用MD、G418-YPD平板和PCR法筛选Mut 表型,获得了分泌表达的重组毕赤酵母。随机挑取一菌株作为表达菌,用甲醇诱导PeaT2蛋白表达。SDS-PAGE及Western blot检测结果均表明PeaT2在毕赤酵母中成功地分泌表达。用peaT2基因的表达蛋白处理小麦种子,生物测定表明,表达蛋白能明显促进小麦的生长,具有蛋白激发子作用。     

新疆家蚕抗菌肽在毕赤酵母中表达及活性研究

目的:在毕赤酵母中表达新疆家蚕抗菌肽基因(Cecropin-XJ)并检测其活性.方法:根据作者实验室已克隆获得的新疆家蚕抗菌肽(Cecropin-XJ)基因设计引物,通过PCR方法扩增Cecropin-XJ,将PCR产物和表达载体pPIC9K用EcoR Ⅰ及Not Ⅰ双酶切,构建重组表达质粒pPIC9K-(Cecropin-XJ),酶切及测序正确后,电转化到毕赤酵母GS115,对分泌表达的重组蛋白进行活性检测.结果:PCR扩增获得192 bp Cecropin-XJ,成功构建pPIC9K-Cecropin-XJ,优化诱导条件证明在pH 6的BMMY培养液中,0.5%甲醇诱导约48h后,获得的表达产物活性较强,对多种革兰氏阴性菌和阳性菌具有抗菌活性,在100℃条件下,其活性可维持100min以上.结论:新疆家蚕抗菌肽在毕赤酵母中分泌表达,为大规模发酵生产奠定了基础.     

人粒细胞-巨噬细胞集落刺激因子的克隆及在毕赤酵母中的表达

采用加长引物5＇端的方法克隆了hGM-CSF的编码基因,克隆过程中对该基因的局部做了密码子优化。然后克隆进毕赤酵母分泌型表达载体pPIC9K,电击转化毕赤酵母。PCR、SDS-PAGE与Western blotting证实hGM-CSF已整合进酵母基因组,摇瓶水平粗蛋白表达量达389mg/L,动物实验证实蛋白产物活性正常,SDS-PAGE与N-糖苷酶F去糖基化分析发现hGM-CSF被糖基化。     

一种新型抗菌肽Cecropin B真核表达载体的构建

通过重叠区扩增法人工设计和合成一种新型抗菌肽Cecropin B基因,按正确的阅读框架定向克隆至巴斯德毕赤酵母的高效表达载体pPIC9K上。经PCR鉴定及序列分析,所转化的大肠杆菌DH5ct菌落中含有插入Cecropin B基因的重组质粒pPIC9K—CB,表明成功构建了新型抗菌肽Cecropin B表达载体pPIC9K—CB。     

抗菌肽牛乳铁多肽素（LfcinB）在毕赤酵母中的表达及活性鉴定

利用巴斯德毕赤酵母(Pichia pastors)系统表达抗菌肽——牛乳铁多肽素(bovine lactoferricin,简称Lf-cinB),获得的分泌型表达产物具有较强的抗菌活性。首先将人工合成的LfcinB基因片段克隆到巴斯德毕赤酵母分泌型表达载体pPIC9K中,获得的重组质粒pPIC9K-LfcinB通过限制性内切酶SalⅠ酶切线性化,经电穿孔法转化入毕赤酵母细胞SMD1168内。G418抗性筛选,得到高拷贝转化子,经PCR检测LfcinB基因与毕赤酵母染色体稳定整合。阳性克隆经甲醇诱导表达LfcinB。结果表明,抗菌肽牛乳铁多肽素基因已经整合到酵母细胞基因组中并获得表达,表达产物具有较强的杀菌作用。     

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.     

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.     

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.