北京地区人呼吸道合胞病毒核蛋白基因的序列分析及原核表达

为了解北京地区流行的人呼吸道合胞病毒(HRSV)N蛋白基因的特征,并用大肠杆菌表达获得N蛋白,从2006年1月至3月收集的首都儿科研究所附属儿童医院急性呼吸道感染患儿标本中分离到7株HRSV毒株(3株A亚型,4株B亚型),分别经RT-PCR扩增得到HRSV N蛋白全基因,克隆至pUCm-T载体中并进行测序和分析;从重组质粒pUCm-N9968中经PCR扩增获得N基因,亚克隆入原核表达载体pET30a( )中,得到重组表达质粒pET30a-N9968,转化大肠杆菌BL21(DE3),IPTG诱导培养;SDS-PAGE和Western blot检测目的蛋白的表达和抗原性。经扩增并测序,7株HRSV N蛋白基因全长均为1 176 bp,编码一个含391个氨基酸的蛋白;北京地区7株RSV分离株的N蛋白基因之间的核苷酸和推导的氨基酸同源性分别在85.4%~99.7%之间和95.4%~100%之间,进一步证明N蛋白基因的高度保守性。经诱导培养产生大量带6个组氨酸标记的重组N蛋白,主要以包涵体形式存在。N蛋白粗提物经Ni2 亲和层析获得较理想纯化。Western blot结果显示,所表达的蛋白能与特异性单克隆抗体和人血清反应。说明本研究构建的重组pET30a-N9968原核表达质粒使N蛋白获得了高效表达,并且具有特异的抗原活性,为今后进一步研究奠定了基础。     

猪源戊型肝炎病毒基因IV型ORF3编码蛋白的表达及鉴定

通过PCR从已构建的猪源戊型肝炎病毒全基因克隆扩增ORF3全基因,将扩增产物插入到pMD18-T载体中,亚克隆至原核表达载体pET28a（+）,构建pET28a-ORF3表达载体,转入E.coli BL21 (DE3),IPTG诱导表达。Ni-NTA层析柱纯化表达蛋白,用SDS-PAGE、免疫印迹、ELISA等方法分析鉴定表达产物。结果成功扩增到345 bp的目的基因;构建了重组表达载体pET28a-ORF3;转化宿主菌E.coli BL21 (DE3)后表达产物的相对分子质量在6.50~16.5 kDa之间,与预期表达的目的蛋白相对分子质量相符;表达的目的蛋白能与阳性猪源和人源血清发生特异性反应,证实其具有较好的反应原性。     

猪链球菌2型溶血素基因与38KDa蛋白基因克隆及联合表达

目的:研究有效的猪链球菌病疫苗。方法:以四川株猪链球菌2型基因组DNA为模板分别扩增溶血素基因和38KDa基因主要功能区基因;并经连接、克隆及酶切鉴定。分别构建原核表达载体pET32a-Sly、pET32a-38KDa,提取阳性克隆质粒分别进行双酶切并纯化,通过PCR串联两片断,将目的片断定向克隆到表达载体pET32a中,经测序正确后,重组质粒转化入大肠杆菌BL21 (DE3),用IPTG进行诱导表达。结果:重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子量约为60Ku的重组蛋白,表达产物经纯化后,免疫印迹法(Western-blotting)证实该重组蛋白可以与SS2阳性血清发生特异性反应。结论:本研究为重组蛋白的免疫保护应用奠定基础。     

人β神经生长因子基因稀有密码子及mRNA二级结构对其在大肠杆菌中表达的影响

目的:研究人β神经生长因子(β-NGF)基因中稀有密码子及其mRNA二级结构对其在大肠杆菌中表达量的影响。方法:根据对人β-ngf中稀有密码子及其mRNA二级结构的研究,同义突变人β-ngf基因,通过PCR得到人β-ngf的5’端同义突变基因rh-β-ngfp32和全同义突变基因rh-β-ngfmu,将这2个序列克隆入载体pET3a中,得到重组质粒pET3a-ngfp32和pET3a-ngfmu,分别转化大肠杆菌BL21(DE3)感受态,IPTG诱导表达,收集菌体,SDS-PAGE检测其表达量的改变。结果:构建的pET3a-ngfp32和pET3a-ngfmu表达载体酶切和测序结果正确,SDS-PAGE结果显示,与在重组菌pET3a-NGF总蛋白中的表达量相比,目的蛋白rh-β-NGF在重组菌pET3a-NGFP32和pET3a-NGFmu中的表达量均明显增高,并且在重组菌pET3a-NGFmu中的表达量高于重组菌pET3a-NGFP32。结论:目的蛋白rh-β-NGF在重组菌pET3a-NGFP32和pET3a-NGFmu中表达量的增高,说明人β-ngf基因中稀有密码子和mRNA的二级结构对其在大肠杆菌中的表达有较为明显的影响,结果为构建rh-β-NGF的大肠杆菌工程菌株奠定了基础。     

伤寒沙门菌线性质粒pBSSB1的p17基因的生物学功能分析

H:z66阳性伤寒沙门菌含有一特有的介导鞭毛单向相变换的线性质粒pBSSB1,其上第17号基因(p17)编码一未知功能蛋白(P17)。利用λ-Red系统制备p17基因缺陷变异株,测定变异株和野生株的生长曲线,比较其生长差异,同时通过半固体LB培养基进行动力试验比较其动力差异。利用PCR技术扩增获得p17基因,构建其重组表达载体pET28a(+)-p17,通过镍柱纯化目的蛋白P17-His6,纯化后蛋白作为抗原免疫兔子以制备多克隆抗体。结果显示,制备了p17基因缺陷变异株,变异株的生长明显迟缓于野生株(P0.05),变异株的动力明显减弱。构建了重组表达载体pET28a(+)-p17,纯化获得了高纯度的目的蛋白P17-His6并制得相应的多克隆抗体。     

Gluconobacter oxydans WD膜结合山梨醇脱氢酶基因在E.coli中的克隆表达

本研究构建了四株含有氧化葡萄糖酸杆菌山梨醇脱氢酶基因的重组大肠杆菌,并初步探究SldB和SldA亚基在山梨醇脱氢酶转化甘油反应中的作用。将pET28a、pETduet与PCR扩增的目的基因连接,构建单启动子调控重组质粒pET28a-sldB、pET28a-sldA、pET28a-sldBA和双启动子调控重组质粒pETduet-sldB'-sldA'。只有含pET28a-sldBA和pETduet-sldB'-sldA'的重组菌具有转化甘油的活性,表明G.oxydans WD的山梨醇脱氢酶催化甘油脱氢需要SldB和SldA亚基的共同作用。串联基因sldBA的蛋白表达结果与双启动子控制sldB和sldA基因蛋白表达结果基本相同,表明位于sldB基因末端的sldA的RBS序列可被E.coli C43的核糖体识别。     

含CSFV E2基因A/D区原核表达载体的构建、表达及其抗原性研究

应用RT PCR方法扩增了编码猪瘟病毒石门株 (CSFVshimenstrain)囊膜糖蛋白E2全基因 ,然后将其克隆到pMD 1 8T质粒中 ,获得重组质粒pMD E2。再以pMD E2为模板 ,另行设计两对引物 ,同时扩增其中一段适于在E .coli中表达且抗原反应性较好的基因片段 (E2蛋白A D抗原区基因序列 ) ,将扩增的两片段串联插入原核表达载体pET 32a中构建成重组质粒pET 2e。用酶切和序列分析鉴定插入目的基因的正确性。SDS PAGE和Western blot分析表明 ,经pET 2e转化、IPTG诱导的受体菌可表达目的蛋白 ,克隆在硫氧还蛋白 (thioredoxinprotein ,TrxA)基因下游的E2蛋白基因与TrxA基因获得了高效融合表达 ,并且具有免疫学反应活性 ,这为猪瘟的血清学诊断方法的建立打下了基础 。     

牛分枝杆菌mpb64基因的克隆、鉴定及其表达

以牛型分枝杆菌基因组DNA为模板,PCR方法扩增mpb64基因,纯化PCR产物并与pDM18-T载体连接、转化,经酶切及核苷酸序列鉴定为正确后,酶切产物亚克隆到原核表达载体pET30a(+)的KpnI／EcoRI位点,构建重组表达质粒pET30a+-mpb64,转化到大肠杆菌DE3内,以IPTG进行诱导,终浓度为1mmol／L,诱导产物进行SDS-PAGE电泳。结果表明,PCR方法成功扩增出mpb64基因,核苷酸序列测定验证了其正确性,重组表达质粒表达的pET30a+-mpb64融合蛋白相对分子量为30.4kDa,与实测相符。牛分枝杆菌pET30a+-mpb64的成功表达为牛结核病的诊断及新型疫苗的研究奠定了基础。     

丝状支原体丝状亚种SC型脂蛋白Q N末端基因的表达、纯化与抗原性分析

为了表达丝状支原体丝状亚种SC型(MmmSC)中国分离株HVRIⅩ脂蛋白Q(LppQ)N末端基因,将该基因经PCR扩增后克隆至原核表达载体pET32a中,经酶切、PCR、测序证实获得了重组表达质粒,转化Escherichia coliBL21(DE3)菌,经IPTG诱导后获得可溶性融合蛋白,表达量占菌体总蛋白的53.7%,用Ni-NTAHis.Bind纯化试剂盒纯化后,蛋白纯度达95%以上。表达蛋白经Western blot检测其抗原活性,结果表明纯化蛋白可与CBPP标准阳性血清发生强烈的反应,而与阴性血清不发生反应。     

立氏立克次体外膜蛋白H 基因片段的克隆表达与免疫原性分析

目的:克隆表达立氏立克次体(Rickettsia rickettsii)外膜蛋白H基因(ompH)片段并对其进行免疫原性分析。方法:采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果:获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论:pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

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.     

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.     

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.