融合表达载体pET32a/Trx-EK-MrVIB的构建及在大肠杆菌中表达

人工设计合成芋螺毒素基因Mr VIB来构建表达载体p ET32a/Trx-EK-Mr VIB,将其转化大肠杆菌BL21(DE3)plys S进行诱导表达。菌体经超声破碎后利用Ni-NTA琼脂糖柱进行亲和层析纯化融合蛋白,SDS-PAGE电泳分析融合蛋白表达。结果表明融合表达载体p ET32a/Trx-EK-Mr VIB经PCR扩增和测序鉴定具有正确的开放阅读框。SDS-PAGE电泳显示融合蛋白在大肠杆菌中获得高效可溶性表达,经一步亲和层析获得纯度大于90%的融合芋螺毒素达73.6 mg/L。本文成功构建了融合表达载体p ET32a/Trx-EK-Mr VIB,融合芋螺毒素Trx-EK-Mr VIB在大肠杆菌中获得高效可溶性表达。     

大肠杆菌yfiF基因原核表达系统构建、表达条件优化及蛋白纯化

[目的]构建大肠杆菌功能未知基因yfi F的原核表达系统,对诱导表达条件进行优化,并纯化表达的可溶性Yfi F融合蛋白。[方法]使用p ET16b表达质粒构建p ET16b-yfi F原核表达载体,转化大肠杆菌BL21,IPTG诱导表达Yfi F融合蛋白,对IPTG加入时机、终浓度及诱导时间进行优化,并使用镍柱纯化裂菌上清液中的可溶性Yfi F融合蛋白。[结果]构建了p ET16b-yfi F重组质粒,IPTG诱导表达Yfi F融合蛋白,最佳诱导条件为细菌生长至OD600值为1时加入IPTG,终浓度为0.1 mmol/L,诱导9 h。裂菌上清液中的可溶性Yfi F融合蛋白纯化后浓度为209μg/ml。[结论]成功构建了yfi F的原核表达系统,优化了诱导表达条件,可溶性Yfi F融合蛋白得到纯化。     

人纤溶酶原饼环区5(hPK5)基因的分泌型表达

构建人纤溶酶原饼环区5(hPK5)基因的原核可溶性表达载体并进行表达和纯化,获取大量高纯度、具有生物活性的hPK5蛋白。以纤溶酶原cDNA为模板,PCR扩增了hPK5基因,经过适当酶切后构建表达载体pET22b(+)hPK5,转入大肠杆菌BL21(DE3)进行表达并经组氨酸亲和层析获得纯化。带有重组质粒pET22b(+)hPK5的大肠杆菌经IPTG诱导后以可溶性形式表达16kDa的蛋白,其表达量占菌体总蛋白的30%以上,纯化后目的蛋白纯度达95%以上,Western印迹表明重组蛋白具有Histag抗原活性。构建了pET22b(+)hPK5重组质粒并成功地在大肠杆菌中获得可溶性表达,为获得大量hPK5基因工程产品奠定了实验基础。     

重组芋螺毒素His-Xa-MrVIB分泌表达研究

[目的]利用简单快速的基因工程法来生产富含二硫键的芋螺毒素Mr VIB,寻找有效合成具有天然活性芋螺毒素的新途径。[方法]人工设计合成芋螺毒素Mr VIB基因引物来构建表达载体p ET22b(+)/His-Xa-Mr VIB,将其转化大肠杆菌BL21(DE3)plys S进行诱导表达。再利用Ni-NTA琼脂糖柱进行亲和层析纯化重组蛋白,Tricine-SDS-PAGE电泳分析重组蛋白表达形式。[结果]重组芋螺毒素His-Xa-Mr VIB(r His-Xa-Mr VIB)在大肠杆菌中获得有效分泌表达,经一步亲和层析获得纯度大于90%的重组芋螺毒素。[结论]基因工程方法能够有效分泌表达芋螺毒素Mr VIB,解决化学合成芋螺毒素产量低、成本高、难以纯化等问题。     

红鳍东方鲀(Takifugu rubripes)干扰素调节因子2基因的克隆及原核表达

干扰素调节因子(Interferon regulatory factor,IRF)是一种多功能的转录因子。采用反转录聚合酶链式反应(RT-PCR)和c DNA末端快速扩增(RACE)技术克隆了红鳍东方鲀干扰素调节因子2(Interferon regulatory factor 2,IRF2)基因的全长c DNA。该基因全长为1 552 bp,其中5'非编码区(5'-UTR)187 bp,3'非编码区(3'-UTR)429 bp,编码区(CDS)为936 bp,共编码311个氨基酸。将IRF2的编码区序列连接到原核表达载体p ET32a(+),成功构建了重组体p ET32a(+)-IRF2。将重组体p ET32a(+)-IRF2转入大肠杆菌感受态细胞BL21(DE3)中,获得了重组表达IRF2的基因工程菌。经IPTG诱导,p ET32a(+)-IRF2在BL21中得到了融合表达。通过对表达产物的纯化和Western blotting检测,结果显示红鳍东方鲀IRF2基因在大肠杆菌中的表达效果较高,目的蛋白准确。     

大肠杆菌trpBA基因的克隆表达

目的:提高大肠杆菌中色氨酸合成酶的表达量和表达活性。方法:利用PCR方法从大肠杆菌K-12的基因组中直接克隆出紧密连锁trpB和trpA基因(简称trpBA),并将其连接到原核表达载体pet22b( )中,得到重组质粒pet22b( )-trp-BA,转化大肠杆菌BL21,IPTG诱导重组蛋白表达,表达产物经SDS-PAGE分析并用比色法测定其活性。结果:凝胶电泳可见PCR扩增产物大小约为2kb,SDS-PAGE鉴定目的蛋白的Mr分别约为29000和44000,色氨酸合成酶α、β亚基分别得到了高效表达,色氨酸合成酶活性提高到对照菌的3.7倍。结论:成功构建了重组质粒pet22b( )-trpBA,色氨酸合成酶的表达量和表达活性在大肠杆菌中得到了提高,为高产色氨酸基因工程菌的构建奠定基础。     

解淀粉芽孢杆菌TF28抗菌蛋白基因TasA克隆与表达

[目的]克隆解淀粉芽孢杆菌TF28抗菌蛋白基因Tas A并进行原核表达和抑菌活性研究。[方法]采用PCR方法扩增抗菌蛋白基因Tas A,连接p ET22b载体,导入E.coli BL21(DE3)菌株,进行IPTG低温诱导表达,用His柱纯化表达产物,采用纸片方法测定其抑菌活性。[结果]从解淀粉芽孢杆菌TF28中克隆了抗菌蛋白基因Tas A,以p ET22b为表达载体构建高效表达抗菌蛋白Tas A的基因工程菌株,该菌株在0.05 mmol/L IPTG 15℃诱导4 h,Tas A蛋白表达率为34.2%,经His柱纯化后获得SDS-PAGE电泳一条带的纯化Tas A蛋白,其含量为67.8 mg/L,收率为90.8%。该蛋白抑制番茄灰霉病和叶霉病、玉米茎基腐病和水稻稻曲病菌生长。[结论]实现抗菌蛋白基因Tas A的原核表达,表达率34.2%,表达蛋白具有广谱抑菌活性,在植病生防方面具有应用潜力。     

重组蛋白ES-Kringle5的表达、纯化及活性检测

目的：利用基因工程方法原核表达重组融合蛋白ES-Kringle5并进行纯化及活性检测。方法：ES-Kringle5是将内皮抑素N端的前27个氨基酸与Kringle5通过连接肽相连的重组融合蛋白,合成该重组蛋白的基因片段并插入载体pMD18-T中,然后克隆至大肠杆菌表达载体pET25b中并转化E.coli BL21（DE3）。乳糖诱导表达后经Ni-NTA亲和层析纯化后获得目的蛋白。通过抑制HUVEC细胞增殖实验检测其生物学活性。结果：重组质粒构建正确。利用乳糖诱导表达并降低诱导温度能增加目的蛋白的产量及可溶性表达。纯化后的重组蛋白纯度大于95%。生物学活性证明该重组蛋白具有抑制HUVEC的增殖能力。结论：具有生物学活性的重组蛋白ES-Kringle5可在大肠杆菌中高效表达,为研究其体内药效、药代及安全性评价奠定了基础。     

拟南芥信号肽AtRALF1的表达、纯化及活性分析

利用RT-PCR扩增获得拟南芥At RALF1基因的全长cDNA序列,将其构建入携带His标签的原核表达载体p ET28b中,获得重组表达载体p ET28b-At RALF1,并将其转入到大肠杆菌BL21(DE3)中。随后在不同的IPTG浓度、温度和诱导时间条件下,进行At RALF1蛋白的表达研究,建立了At RALF1融合蛋白的高效表达体系。结果表明,在30℃、1 mmol/L IPTG的条件下诱导4 h,At RALF1融合蛋白的表达量最大。进一步用获得的At RALF1融合蛋白处理苗龄5 d的野生型拟南芥(Col-0)植株,发现其根的生长受到了抑制,表明我们获得了具有活性的At RALF1小肽,为进一步研究该小肽奠定了基础。     

人纤溶酶原kringle 5在大肠杆菌中克隆和分泌表达

构建能够表达分泌性的人纤溶酶原kringle 5（简称hPK-5）的大肠杆菌工程菌。用PCR方法扩增获得hPK-5基因,构建原核表达载体pET-22b（＋）/hPK-5,转化大肠杆菌BL21（DE3）,经IPTG诱导表达并鉴定其免疫学活性。成功表达14kD的重组hPK-5,且约占菌体分泌性蛋白20%以上,经Western Blot分析表明其具有hPK-5的免疫抗原活性。人纤溶酶原kringle 5在大肠杆菌中BL21（DE3）获得可分泌表达。     

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.     

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.     

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.     

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.