单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别。表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白。重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答。     

小鼠IL-35基因毕赤酵母表达载体的构建及表达

目的构建真核酵母表达载体pPIC9K与小鼠IL-35基因的重组质粒pPIC9K-mIL-35-His,在毕赤酵母GS115菌株中诱导表达,并对其进行鉴定。方法以pET-30a-mIL-35为模板,用PCR扩增出IL-35去信号肽基因全序列,并在3'端引入His标签,构建重组质粒pPIC9K-mIL-35-His,经SalⅠ线性化重组质粒后,用电穿孔方法将该基因转染毕赤酵母细胞内。经G418梯度筛选高拷贝转化子,筛选Mut+表型后经PCR进一步鉴定IL-35基因与酵母染色体是否整合。小量诱导表达筛选出高表达菌株,大量甲醇诱导表达,以SDS-PAGE及免疫印迹(Western blot)鉴定蛋白表达。结果小鼠IL-35基因真核酵母表达载体pPIC9K-mIL-35-His构建成功,并且在GS115中通过甲醇诱导表达,经SDS-PAGE及Western blot分析,可见相对分子质量约为65 000的目的蛋白。结论实验所构建的重组质粒pPIC9K-mIL-35-His可在毕赤酵母GS115中正确的表达小鼠IL-35基因。     

OSF1在毕赤酵母菌中的分泌表达及其生物活性

为了研究人成骨细胞刺激因子(Human osteoblast-stimulating factor,OSF-1)的生物学活性,构建OSF-1高效毕赤酵母表达菌株并表达纯化具有生物活性的OSF-1。首先通过全基因人工合成的方法合成人osf-1基因,并克隆至毕赤酵母分泌表达载体pPIC9K,构建重组表达质粒pPIC9K/osf-1,线性化后电转化酵母宿主菌GS115,构建P.pastoris GS115/pPIC9K/osf-1,经MD、G418-YPD平板和PCR法筛选,获得多拷贝转化子。阳性表达菌株在25℃,经1%的甲醇诱导表达96 h,重组蛋白的表达量达到最大。经SDS-PAGE电泳分析所表达的重组蛋白约为18 kDa,与人成骨细胞刺激因子相符。表达上清经SP-Sephadex C-50离子交换层析进行纯化,得到纯度达98%以上的OSF-1。Western blotting鉴定该蛋白对人成骨细胞刺激因子抗体具有良好的抗原性。生物活性测定表明提纯的OSF-1能促进鼠成骨细胞MC3T3-E1的增殖和分化。利用重组毕赤酵母高效分泌表达了有生物活性的OSF-1,为进一步开展其抗骨质疏松活性研究及产业化开发奠定了基础。     

黑曲霉果胶裂解酶基因毕赤酵母在Pichia pastoris GS115中的表达

利用RT-PCR技术从黑曲霉(EIM-6)中扩增得到去除信号肽的果胶裂解酶基因A,将其插入到毕赤酵母表达载体pPIC9k上,构建重组表达质粒pPIC9K-pelA,电击转化毕赤酵母GS115,得到了表达成功的工程菌株。用终浓度为1.5%的甲醇对其进行诱导,将发酵上清液浓缩后,用盐酸法测定其酶活可以达到2.3U/mL。通过对重组毕赤酵母诱导表达产物进行SDS-PAGE鉴定,发现重组毕赤酵母分泌了1个约38kD的蛋白,与该酶基因产物的理论值相符,并通过水解圈法测定验证,均说明果胶裂解酶得到正确的分泌表达.     

极细链格孢菌peaT1基因在毕赤酵母中的表达与功能 分析

建立了在毕赤酵母(Pichiapastoris)中分泌表达PeaT1蛋白的技术。将来源于极细链格孢菌(Alternaria tenuissima)的基因peaT1亚克隆至酵母分泌型表达载体pPIC9K,构建了重组表达载体pPIC9K-peaT1,分别用SalI或BglII酶切线性化后电击转入毕赤酵母GS115菌株中,经MD平板筛选、PCR鉴定获得整合有外源基因的重组菌株。在α-Factor及AOX1基因启动子和终止信号的调控下,PeaT1在酵母中大量表达并分泌到胞外,SDS-PAGE检测表明表达蛋白的表观分子量约为35kD。表达蛋白上清稀释液能诱导烟草产生对TMV的抗性,其枯斑数抑制率可达到30.37%。每升表达上清液经超滤浓缩和离子交换层析可纯化目的蛋白16.13mg,该纯化蛋白能显著地促进小麦幼苗的生长。     

钙网蛋白-N58在毕赤酵母中的表达及活性分析

目的：利用毕赤酵母表达系统表达Calreticulin-N58蛋白。方法：利用重叠延伸PCR方法合成Calreticulin-N58基因,构建成分泌型的重组表达载体pPIC9K-CRT-N58。该重组表达载体经SacI线性化后,电激转化入毕赤酵母GSll5,经MD板和不同浓度G418筛选得到多拷贝重组菌株。经甲醇诱导表达目的蛋白,SDS-PAGE分析重组蛋白的表达情况,在鸡胚中进行活性分析。结果：重组质粒pPIC9K-CRT-N58在毕赤酵母中经甲醇诱导能分泌表达CRT-N58蛋白,摇瓶表达量大约为60mg／L,纯化的目的蛋白有显著的血管生成抑制作用。结论：实验成功地在毕赤酵母表达系统中实现了Calreticulin-N58的分泌表达。为Calreticulin-N58蛋白的进一步药用研究奠定了基础。     

人甲状旁腺激素(1-34)二联体与人血清白蛋白融合蛋白的构建及表达

构建人甲状旁腺激素(1-34)二联体与人血清白蛋白融合蛋白的表达载体,并表达得到该融合蛋白.通过设计强特异性的引物,利用重叠PCR技术,定向定量的拼接得到hPTH(1-34)二联体-HSA融合蛋白的基因;将构建好的融合基因插入表达载体pPIC9K,大量扩增重组质粒,并用Sal I线性化,电击转化毕赤酵母GS115,经组氨酸缺陷和G418抗性双重筛选得到阳性转化子;挑选阳性转化子进行甲醇诱导表达.测序结果表明得到的重组质粒pPIC9K-hPTH(1-34)二联体-HSA与目标设计完全一致;基因组PCR鉴定结果证明成功构建了hPTH(1-34)二联体-HSA融合基因的毕赤酵母(GS115)表达系统;SDS-PAGE电泳表明融合蛋白获得了表达,尿微量白蛋白试剂盒测定甲醇诱导表达3d后融合蛋白的产量为127 mg/L.     

柔嫩艾美耳球虫钙依赖蛋白激酶3基因在毕赤酵母中的表达及分析

旨在真核表达系统中高效表达柔嫩艾美耳球虫钙依赖蛋白激酶3(Eimeria tenella calcium-dependent protein kinase-3,EtCDPK3),获得有活性的天然蛋白,利用毕赤酵母表达系统对该基因进行了表达.将EtCDPK3基因连接到毕赤酵母表达载体pPIC9K上,构建重组质粒pPIC9K-EtCDPK3.重组质粒通过电击转化入酵母细胞GS115后,用组氨酸缺陷培养基和G418分别进行筛选,获得含重组质粒的酵母表达细胞.重组酵母细胞在含1％甲醇的BMMY培养基中诱导产生目的蛋白,培养收集1-4d的部分上清.经SDS-PAGE检测,所表达的蛋白相对分子质量约为49 kD.Western blotting表明,该蛋白能与兔抗EtCDPK3血清特异性结合.结果表明,柔嫩艾美耳球虫CDPK3基因在毕赤酵母中成功地进行了表达.     

内切葡聚糖酶基因在大肠杆菌与毕赤酵母中的表达

根据绿色木霉(Trichoderma viride)WL 0422菌株内切葡聚糖酶基因egⅡ的cDNA序列,设计特异引物,以重组质粒pTG19-T-egⅡ为模板,扩增得到全长1 194bp的egⅡ成熟肽cDNA片段.将该片段分别克隆到大肠杆菌(Escherichia coil)表达载体pET-28a( )和毕赤酵母(Pichia pastoris)表达载体pPIC9K中,并在大肠杆菌Rosset(DE3)和毕赤酵母GS115中进行了表达.重组大肠杆菌表达蛋白占菌体总蛋白的15%,表达产物无内切葡聚糖酶活性.重组毕赤酵母经甲醇诱导实现了分泌表达,在摇床水平上酶活性达到2 788U/ml.酶学性质分析表明,该酶作用的最适pH为4.5,pH 3.5～6.0范围内酶活性保留在80%以上;最适温度为50℃,55℃以下酶的稳定性在90%以上.     

昆虫神经毒素LqhIT2的表达、抗血清制备及活性分析

根据毕赤酵母密码子偏爱性,不改变毒素蛋白质一级结构,设计合成了昆虫神经毒素LqhIT2基因,并分别克隆至大肠杆菌融合表达载体pPET30-a( )和毕赤酵母分泌表达载体pPIC9K.在IPTG的诱导下,神经毒素在大肠杆菌中融合表达,表达产物经镍亲和层析纯化后,用于免疫BALB/c小鼠,制备了特异性较高的抗血清,抗体滴度超过1:128 000.利用制备的抗血清,采用斑点杂交,筛选得到了较高水平分泌表达重组LqhIT2的酵母转化子,摇瓶条件下,毒素表达量约9 mg/L.大肠杆菌表达产物没有生物活性,酵母表达产物经注射蝗虫表现出杀虫活性.     

Tuning of electronic properties of one-dimensional cyano-bridged Cu-Ni, Cu-Pd, and Cu-Pt bimetallic assemblies by stereochemistry of ligands

Preparations, XPS and electronic spectroscopy, and magnetism of seven new one-dimensional cyano-bridged coordination polymers, chiral [Cu(RR-chxn)₂][Pd(CN)₄] · 2H₂O (1), [Cu(trans-chxn)₂][M(CN)₄] · 2H₂O (2, 4, and 6 for M = Pd, Ni, and Pt), and [Cu(cis-chxn)₂][M(CN)₄] · 2H₂O (3, 5, and 7 for M = Pd, Ni, and Pt) (RR-chxn = cyclohexane-(1R,2R)-diamine, trans-chxn = racemic trans-cyclohexane-(1,2)-diamine, and cis-chxn = racemic cis-cyclohexane-(1,2)-diamine) have been reported in view of tuning of their electronic properties by stereochemistry of chxn ligands and metal-substitution. Comparison of Cu 2p_1/2 and 2p_3/2 peaks of XPS and broad d-d bands around 18 000 cm⁻¹ of electronic spectra are described systematically for 1-7. Variable-temperature magnetic measurement shows that complexes 1-7 indicate weak antiferromagnetic interactions via cyano-bridges. Because of semi-coordination coupled with pseudo Jahn-Teller elongation and electrostatic interaction for 1, the axial Cu-N coordination bond distances of 2.330(7) and 3.092(8) Å are considerably longer than those of equatorial ones in the range from 2.016(6) to 2.030(6) Å. The former bond distances of 1 are intermediate values among the related Ni (2.324(6) and 3.120(8) Å) and Pt (2.34(1) and 3.09(1) Å) complexes.     

Land,livestock, and livelihoods: Changing dynamics of gender,caste, and ethnicity in a Nepalese Village

Over the past 10 years, Ghusel VDC, Lalitpur District has moved from primarily subsistence agriculture into the wider cash economy aided by the Small Farmers' Development Program (SFDP), which provides credit to farmers mainly for the purchase of buffalo for milk production, and by the National Dairy Corporation, which supports local dairy cooperatives. Analysis reveals that buffalo-keeping and milk sales are increasing the well-being of many households, while at the same time creating new inequalities in gender roles and responsibilities, greater inequities between Brahmin and Tamang residents in Ghusel, and placing pressures on the ecosystem for increased supplies of fodder and fuelwood. Evidence suggests that there is critical, need for attention to the social, and particularly gender-based, implications of maintaining livestock for milk sales and to the ecological underpinnings of this livelihood system.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Chiral Separation of G‐type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G‐type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical‐scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(–) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(–) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. Chirality 26:817–824, 2014. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc.     

Influence of Fluoride on Rat Kidney Antioxidant System: Effects of Methionine and Vitamin E

The aim of the study has been to determine and compare the influence upon the kidney antioxidative system, exercised by administration of vitamin E, and vitamin E in combination with methionine, under conditions of oxidative stress induced by sodium fluoride. The experiment was carried out on Wistar FL rats (adult males) that, for 35 days, were administered water, NaF, NaF with vitamin E, or vitamin E with methionine (doses: 10 mg NaF/kg of body mass/24 h, 3 mg vitamin E per 10 μl per rat for 24 h, 2 mg methionine per rat for 24 h). The influence of administered sodium fluoride and antioxidants upon the antioxidative system in kidney was examined by analyzing the concentration of malondialdehyde (MDA) and the activity of the most important antioxidative enzymes (SOD, total and both its isoenzymes, GPX, GST, GR, and CAT). The studies carried out confirmed the disadvantageous effect of the administered dose of NaF upon the antixodiative system in rats (increase in the concentration MDA, decrease activity of all antioxidative enzymes). The administration of vitamin E increased the activity of studied enzymes with the exception of glutathione reductase GR; it also reduced the procesess of lipid peroxidation. It has been found that combined doses of vitamin E and methionine were most effective in inhibiting lipid peroxidation processes. The results confirmed the antioxidative properties of methionine.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

八种脑-肠肽侧脑室内注射对大鼠基础胃酸分泌的影响

用乌拉坦麻醉大鼠作急性实验,采用连续灌流胃并收集流出液的方法,观察向侧脑室内注射微量脑-肠肽对大鼠基础胃酸分泌的影响。实验结果如下:(1)雨蛙肽、八肽胆囊收缩素、促甲状腺素释放激素及四肽胃泌素均使总酸排出量增加;(2)生长抑素、胰多肽、P 物质、胰高血糖素则使总酸排出量减少;(3)上述肽类用侧脑室注射的剂量作肌肉注射,除四肽胃泌素也产生明显的刺激胃酸分泌作用外,对胃酸分泌均无明显影响。以上结果提示,脑内的一些肽类可能以神经递质或调制物的方式,参与中枢对胃酸分泌的调节。     

邻苯二甲酸二丁酯对翡翠贻贝抗氧化酶及脂质过氧化水平的影响

实验室条件下,研究了不同浓度邻苯二甲酸二丁酯(DBP)长期胁迫(15 d)对翡翠贻贝内脏团和外套膜抗氧化酶(超氧化物歧化酶SOD、过氧化氢酶CAT)及脂质过氧化(LPO)水平(以MDA含量表示)的影响,以及受胁迫翡翠贻贝在清洁海水中恢复阶段上述生化指标的变化特征.结果表明:胁迫阶段,0.5和2.5 mg.L-1DBP下翡翠贻贝内脏团SOD活性表现为先抑制后逐渐恢复,12.5和62.5 mg.L-1下则持续受到显著抑制;不同浓度组CAT活性均明显被抑制.LPO水平明显升高.外套膜中,2.5 mg.L-1下SOD活性受到持续诱导,其他浓度组则先被抑制,后随曝露时间延长逐渐被诱导;各浓度组CAT的变化波动较大,没有明显规律;而LPO水平明显升高.净化恢复阶段,12.5和62.5 mg.L-1DBP胁迫下的内脏团SOD和CAT活性恢复较慢,其LPO水平随时间延长逐渐恢复至对照组水平;外套膜中SOD活性呈持续升高趋势,CAT活性和LPO水平则随时间延长恢复到对照组水平.