表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

为进行高密度发酵并实现外源基因的高表达,在表型为Mut^S的重组毕赤酵母（Pichia pastoris）表达人血管生长抑制素的诱导阶段,采用了甘油甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长,又不会发生发酵液中残余甘油及有害代谢产物（乙醇）阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于0.012h^-1,菌体浓度达150 g/L,血管生长抑制素浓度最高达到108 mg/L,血管生长抑制素的平均比生产速率为0.02 mg/(g·h),菌体关于甘油的表观得率为0.69 g/g,菌体关于甲醇的表观得率为0.93g/g,较没有采用甘油限制性流加时都有所提高。     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

甘油及甲醇补料策略对毕赤酵母表达猪α-干扰素的影响

本文研究了甘油及甲醇补料策略对重组毕赤酵母细胞生长及猪α-干扰素(p IFN-α)表达的影响。结果表明,甘油采取不同的补料策略,以及甲醇浓度控制于不同水平时,细胞生长速度不同,p IFN-α抗病毒活性水平也明显不同。高密度发酵阶段,甘油采用指数方式流加控制时,相较于40 g/L/h、10 g/L/h,恒速流加组的细胞浓度最先达最高水平132 g/L,后一直保持稳定,且发酵液中几乎无残留甘油;与之相应的p IFN-α抗病毒活性水平最高。诱导表达阶段,甲醇浓度须控制并恒定于12 g/L时,p IFN-α抗病毒活性最高水平能达到5.95×106IU/m L,而当甲醇浓度稳定于3.5 g/L、16.0 g/L、或者浓度波动较大(10.2~13.8 g/L)时,p IFN-α抗病毒活性均远低于最高水平。     

温度对巴斯德毕赤酵母表达瑞替普酶(reteplase)的影响

研究了温度对巴斯德毕赤酵母表达瑞替普酶(reteplase,rPA)的影响。结果发现,在BMMY摇瓶培养条件下,诱导温度在20℃、25℃,rPA表达量较高,分别是30℃诱导条件下(18.2mg/L)的1.4倍和1.34倍。在高密度发酵过程中,降低诱导温度(20℃、25℃),rPA的表达量在诱导表达84h时达到最高,分别为207.9mg/L和199.5mg/L,比30℃诱导条件下分别提高了35%和30%。通过对细胞活性、蛋白酶和AOX酶活性分析发现,降低温度不仅提高了发酵过程中酵母活细胞率,降低了蛋白酶的活性,减少了rPA的降解,而且提高了AOX酶活,增强了rPA的表达。     

重组巴氏毕赤酵母恒化培养动力学及代谢迁移特性研究

通过对甲醇营养型毕赤酵母基因工程菌以碳源甘油为限制性基质进行恒化培养动力学试验 ,结果认为 :(1 )细胞光密度与其干、湿重呈线性关系 ,当细胞光密度 (OD60 0 )为 1 0 0时细胞湿重 (WCW)为 1 2 8 3g L ,细胞干重 (WDW)则为 2 2 9g L ;(2 )基因工程菌P .pastoris的生长与限制性基质甘油残留浓度的关系符合Monod关系式 ,通过 1 μ对 1 S进行线性回归得 μmax=0 .366h- 1,Ks=0 .1 82 3g L ,经参数推导甘油最大菌体得率系数YG =0 .54g g ,菌体维持生长消耗底物系数m =0 .0 0 69g (g·h) ;氧最大菌体系数YX O2 =30 .96g moL ,菌体维持生长时消耗氧系数mO2 =0 .0 0 0 8mol (g·h) ,最适理论稀释速率Dm =0 .341h- 1;(3)从氨水的消耗速率和呼吸商 (RQ)的变化认为随着比生长速率 (μ)的增大 ,甘油代谢流从糖原异生和磷酸戊糖途径线性地向糖酵解和三羧酸循环途径进行代谢迁移 ,即糖酵解和三羧酸循环途径的代谢流量在线性地增大     

基因重组大肠杆菌表达类人胶原蛋白诱导条件优化研究

为了提高重组大肠杆菌BL21高密度发酵生产类人胶原蛋白的产量,分别研究了诱导时机、乙酸浓度、诱导强度以及补氮方式对外源基因表达的影响,并对各因素进行了优化。采用补料-分批式发酵,初始装液体积为6L。最终细胞密度可达到84.3g／L,类人胶原蛋白的表达量为14.6g/L。结果表明:在对数生长的中后期进行诱导,尽量减少乙酸的积累量,42℃诱导3h后降温至39℃继续诱导,并采用每隔10min补36ml补料氮溶液的周期操作方式,有利于细胞的生长和外源蛋白的表达。     

利用克雷伯肺炎杆菌限制性底物发酵生产1，3-丙二醇

研究了克雷伯肺炎杆菌（Klebsiella pneumoniae）批式流加发酵生产1,3-丙二醇的发酵工艺,根据1,3-丙二醇的生产和菌体生长相关的特点,采用营养基质限制性流加的发酵工艺,通过控制氮源氯化铵以保持细胞稳定生长。结果表明：过低的氮源浓度,细胞生长受到限制,影响产物1,3-PD的合成;过高的氮源浓度,细胞比生长速率增加,但1,3-PD关于消耗甘油的得率降低,用于生长和维持代谢所消耗的甘油量增加。以0.41 g/(L·h)的氮源流加速率,残余氯化铵浓度在0.1 g/L时,转化率和生产强度最高。发酵25 h～28 h后,1,3-丙二醇最终浓度达到52.03 g/L,生产强度为2.04 g/(L·h),相对于甘油的摩尔转化率为0.66,分别比氮源限制前提高了28.0 ％、35.1 ％及29.4 ％。通过限制性流加氯化铵,控制细胞的比生长速率,使底物甘油有效转变为发酵的目标产物1,3-PD,有效实现产物1,3-PD的高生产强度以及对甘油的高转化率。     

比生长速率和氮源对毕赤酵母生产重组鲈鱼生长激素的影响

对毕赤酵母胞内表达重组鲈鱼生长激素(rljGH)的发酵罐上生产进行了研究.建立了指数流加甲醇的策略并考察了不同比生长速率对rljGH生产的影响.结果表明,随着比生长速率的增加,平均比生产速率相应增加,但是胞内持续积累rljGH的时间减少.最大比rljGH产量(0.58 mg/g WCW)在比生长速率为0.029/h时获得.进一步考察了在诱导阶段添加硫酸铵、蛋白胨和酵母抽提物的影响.结果表明,添加硫酸铵和蛋白胨对于rljGH生产没有显著影响;添加2.5 g/L酵母抽提物有助于胞内rljGH的积累,并使胞内积累持续时间由17 h增加到23 h,提高了发酵稳定性.     

通过在低细胞浓度下启动甲醇诱导、优化碳/能量代谢模式促进毕赤酵母表达Monellin

重组毕赤酵母生产表达外源蛋白的过程中,一般在细胞达到高密度后开始启动甲醇诱导。也有报道指出,在较低细胞浓度下,启动甲醇诱导可以有效地控制整个发酵过程的溶解氧浓度,缓解毒副产物的积累,促进目标蛋白的表达。但是,该操作策略下,甲醇/能量调控机制不明,相关研究报道很少。文中以生产表达monellin(甜味蛋白)的重组毕赤酵母为模式菌株,通过在线分析计量甲醇消耗速率、CO2释放速率和O2摄取速率,探讨了不同细胞浓度下启动甲醇诱导和外源蛋白表达体系的甲醇/能量代谢模式。结果表明,在较低细胞浓度(50 g DCW/L)启动诱导并将温度控制在30℃,走向合成monellin前体物质途径的碳流最大(65%),且能与用于ATP再生的碳流形成最佳匹配;monellin的比合成速率与细胞比生长速率完全耦联,且耦联系数最大,比生长速率也较高;理论NADH(能量)利用效率η最高,η在甲醇诱导的绝大部分时段(89%)处于高水平(≥0.8),可以为monellin合成提供足够的能量。因此,该操作条件下,monellin浓度达到2.62 g/L的最高水平,是高细胞密度(100 g DCW/L)启动诱导策略下monellin浓度的2.5-4.9倍。     

运用氨基酸分析法和DOE法优化抗PD-1单克隆抗体生产用培养基

本研究采用氨基酸分析法结合DOE设计法优化并获得高表达抗PD-1单克隆抗体生产用基础和补料培养基。通过对市售多种基础和补料培养基进行筛选,获得细胞生长状况较优的基础培养基和抗体表达较高的补料培养基,利用氨基酸分析法检测较优基础培养基和补料培养基中氨基酸消耗情况,确定影响细胞生长和抗体表达的关键氨基酸种类,利用DOE分析软件设计分别在较优基础和补料培养基中添加不同浓度的氨基酸种类及浓度,根据细胞生长及抗体表达,优化得到抗PD-1单克隆抗体的基础和补料培养基组合。最终优化后基础培养基配方为:Hycell CHO培养基中添加1.04 mmol/L L-天冬酰胺和0.76 mmol/L L-谷氨酰胺。最终优化后补料培养基配方为:OPM CHOCD Feed1补料培养基中添加38.7 mmol/L L-组氨酸,75.0 mmol/L L-酪氨酸,64.0 mmol/L L-丝氨酸,49.2 mmol/L L-谷氨酰胺和18.7 mmol/L L-半胱氨酸。经过3 L反应器培养验证,优化后的培养基比未优化时,最大活细胞密度(PVCD)提高了62.7%,抗PD-1单克隆抗体表达量提高了71.5%,且活性无明显差异。     

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.     

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。     

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.     

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.     

The mechanism of hatching of eggs of Haemonchus contortus

Fluid collected from hatching eggs of Haemonchus contortus contained a lipase which hydrolysed 2-naphthyl laurate (about 0·7 μmol naphthol freed /h/10⁶ eggs). The fluid also hydrolysed l-leucinamide (about 2·3 μmol leucine freed/h/10⁶ eggs). The fluid when added to normal or heated eggs caused ‘hatching’. ‘Hatching’ also occurred in exsheathing fluid from infective juveniles and in a preparation of pancreatic lipase containing leucine aminopeptidase. A purified mammalian leucine aminopeptidase in combination with several different lipases did not attack egg shells.The ‘spontaneous’ hatching of eggs of H. contortus was strongly inhibited by 1,10-phenanthroline, 10^?3M, and this inhibition was reversed by Zn²⁺. However, the inhibition of ‘hatching’ of eggs in externally applied hatching fluid, or the hydrolysis of leucinamide in hatching fluid was generally less marked.     

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.