重组人IL-6/IL-2融合蛋白的高密度发酵和纯化

克隆的IL-6/IL-2融合蛋白编码基因插入pBV220载体,转化BL21(DE3)菌株中表达。发酵过程中,30℃扩增生长6小时,42℃诱导培养4小时,控制溶解氧在30％～50％。发酵液中最终菌体密度(OD600)达29.17(相当于每升发酵液含55克湿菌体),表达的融合蛋白呈包涵体形式。表达蛋白占菌体总蛋白的30％左右。菌体经过超声破碎后反复洗涤包涵体,融合蛋白纯度达到70％,进一步用离子交换层析和凝胶过滤层析纯化使其纯度达95％以上。IL-6/2融合蛋白中的IL-6和IL-2活性分别为1×107和2×105 U/mg。重组人IL-6,/IL-2融合蛋白在大肠杆菌中达到了中试水平的高表达。     

HIV-1核心蛋白的表达与纯化

将编码人I型免疫缺陷病毒(HIV1)核心蛋白p24gag的基因序列克隆到原核表达载体pET28(b)中,高效表达了N,C端融合His·Tagp24蛋白,所表达的重组p24蛋白占菌体总蛋白的46%。在变性条件下,使用NiNTA亲和层析法纯化了p24蛋白,纯度为94%。菌体中及纯化、复性后的目的蛋白均能与抗HIV1p24单克隆抗体发生特异性反应。用纯化的p24蛋白免疫小鼠,4周时小鼠血清抗p24抗体效价达1∶400。实验结果表明:大肠杆菌表达的HIV1p24蛋白纯化后可用作HIV1检测试剂的原料。     

表达rAPC大肠杆菌的高密度发酵及纯化产物的抑瘤活性

重组菌的高密度发酵技术是高效表达异源蛋白的一项重要技术。本文研究了培养基组成。培养条件以及诱导条件等对最终的菌体密度和rAPC表达量的影响，确立了最优的高密度发酵条件。此外，还探讨了不同补料流加方式对菌体生长和rAPC表达的影响，结果表明DO-stat补料流加方式具有显著增加菌体量和重组蛋白表达量的作用。发酵16小时后，菌体密度可达OD600106，每升发酵液中rAPC含量可达3.52g，用纯化好的rAPC对皮下接种S180的小鼠灌胃或腹腔注射，剂量为每天3.4mg/kg-13.4mg/kg，共10天。结果表明rAPC对小鼠S180肉瘤有显著的抑制作用，瘤重抑瘤率分别在45%-64%之间，且对荷瘤小鼠的白细胞数量和胸腺指数均无明显影响。     

番茄乙烯信号转录因子LeERF2在大肠杆菌中的表达和纯化

从破色期番茄果实中提取了总RNA,用RT-PCR方法从中克隆到了627bp全长LeERF2基因.测序结果分析表明,该LeERF2基因序列与已发表序列的同源性为100%.将LeERF2基因亚克隆至载体pET-30a上,构建了原核表达载体pE-LeERF2,将其转化到大肠杆菌BL21中.蛋白质诱导表达的温度为37℃,诱导剂IPTG浓度为1mmol/L,诱导时间为3 h,LeERF2表达蛋白在细菌沉淀中约占菌体总蛋白的58%.应用Ni-NTA亲和层纯化回收的LeERF2表达蛋白的纯度为99%.银染凝胶阻滞实验表明,LeERF2蛋白能与含有GCC盒的逆境相关的几丁质酶启动子基因TLBP1结合.     

鲮生长激素cDNA的克隆及其重组表达产物的促生长活性

利用RT PCR技术从鲮脑垂体组织中克隆出生长激素cDNA片段 ,克隆的mcGH全长 5 6 7bp ,编码由 188个氨基酸残基组成的GH成熟肽。构建了表达载体pQE30 mcGH ,转化大肠杆菌M15 (pREP4 ) ,在初步优化发酵条件的基础上实现了鲮mcGH在大肠杆菌中的高效表达 ,表达的重组蛋白占菌体总蛋白的 32 13% ,经westernblotting分析证实所表达的重组蛋白是mcGH。表达产物主要以包涵体的形式存在。重组蛋白变性、复性后 ,经Ni chelatingSepharose亲和层析纯化 ,SDS PAGE显示 ,纯化的mcGH纯度约为 94 %。纯化的mcGH腹腔注射莫桑比克罗非鱼 (Oreochromismossambicus) ,经 4周处理后 ,实验组 1和 2的体长增长率分别比对照组快 9 38%和 9 75 % ,体重增长率分别快 2 3 4 2 %和 6 2 4 8%。经t检验统计分析表明 ,表达的重组mcGH具有显著的促罗非鱼生长作用 (P <0 0 5 )。     

星突江鲽胰岛素样生长因子I的体外重组表达及生物活性分析

为了在蛋白水平认识星突江鲽(Platichthys stellatus)胰岛素样生长因子I(IGF-I)的生长调控作用及机制,采用RT-PCR方法扩增了其成熟肽片段,利用原核表达载体p ET-28a成功构建了重组星突江鲽IGFI/p ET-28a质粒,转化至大肠杆菌BL21(DE3)后经IPTG诱导获得了N端含6个组氨酸的重组星突江鲽IGFI蛋白。获得的重组IGF-I蛋白大小为12.1 k D,37℃下用0.5 mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)诱导3 h时目的蛋白表达量最高,占菌体总蛋白的39.8%,主要以包涵体形式存在。Western blotting免疫印迹表明星突江鲽IGF-I重组蛋白均可被6×His抗体特异性识别。包涵体经6 mol/L盐酸胍变性、Ni2+离子亲和柱纯化和尿素梯度复性后,可获得高纯度的IGF-I重组蛋白。细胞增殖试验结果显示0.6μg/m L的星突江鲽IGF-I重组蛋白能显著促进人胚胎肾细胞HEK293T的增殖而大于1.8μg/m L时则表现出抑制作用。本研究成功构建了星突江鲽IGF-I体外高效表达系统,并获得具有细胞水平生物活性的星突江鲽IGF-I重组蛋白,结果可为深入探究IGF-I在星突江鲽生长发育中的作用机制及研制高效绿色的促生长制剂提供基础资料。     

真鲷肿瘤坏死因子(TNFα)基因的体外重组与纯化研究

将真鲷肿瘤坏死因子(TNFα)基因的成熟肽区域克隆到表达载体pQE30中,并转化到大肠杆菌XL-blue中进行表达。转化子经载体和基因特异性引物进行PCR双向筛选,并将筛选到的阳性克隆经质粒纯化后,进行序列测定。序列测定结果显示,该基因结构正确,且准确插入到表达载体启动子的下游,获得了结构和组成正确的重组子。重、组子经IPTG诱导后,以SDS-PAGE电泳鉴定,电泳结果表明,该基因在大肠杆菌XL-blue中实现了表达。通过对诱导条件的调整,在体外获得了高效表达的重组蛋白,高效表达的重组蛋白约占菌体蛋白的25％-30％。不同诱导时间对重组蛋白影响实验显示,随诱导时间的增加,重组蛋白产量逐渐增高,经4h诱导,其表达量可以达到平台期,过长时间的诱导,对表达产量没有影响。重组蛋白经镍金属亲和层析纯化,获得了纯度较高的重组蛋白。采用交换缓冲液的方法,用Sephadex G150,对重组蛋白进行脱盐复性,使重组蛋白得到了进一步纯化。     

铜绿微囊藻生物钟蛋白的表达纯化与抗体制备

为了获得融合表达的铜绿微囊藻(Microcystic aeruginosa)生物钟蛋白KaiA、KaiB、KaiC并制备其相应的多克隆抗体,将kaiA、kaiB、kaiC基因分别克隆到原核表达质粒pET-His中.重组质粒pET-His-KaiA,pET-His-KaiB和pET-His-KaiC经酶切和测序鉴定后,分别转化E.coli BL21(DE3)进行融合表达.经SDS-PAGE分析可知,融合表达的KaiA、KaiB和KaiC蛋白表达量可分别达到菌体总蛋白的25%、40%和20%.经亲和层析后融合蛋白KaiA和KaiB的纯度分别达95%和92%,而KaiC经胶回收纯化后纯度也可达93%.将纯化后的三种Kai蛋白作为抗原分别免疫小鼠制备多克隆抗体,经ELISA检测抗体滴度表明,制备的抗KaiA、抗KaiB和抗KaiC的多克隆抗体效价高,分别可达到1:50000、1:60000和1:100000.Western blotting结果表明:获得的多克隆抗体具有较高的效价,抗体能识别相应的Kai蛋白,具有较高的特异性,能用于铜绿微囊藻生物钟蛋白KaiA、KaiB和KaiC的表达节律检测.     

高纯度白喉毒素突变体CRM197的制备及鉴定

为了制备高纯度的白喉毒素无毒突变体CRM197,可作为载体蛋白用于细菌性结合疫苗开发,对CRM197基因进行大肠杆菌密码子优化,并克隆至表达载体pET28a(+）中,将鉴定正确的重组质粒pET28a-CRM197转化到大肠杆菌BL21(DE3),经IPTG诱导表达,并分析其表达形式及表达条件的优化。再对表达的重组CRM197蛋白进行纯化,最后对纯化的CRM197进行WB、纯度、分子量和圆二色谱等检测项目的初步鉴定分析。PCR酶切和测序鉴定结果表明重组质粒pET28a-CRM197构建成功,将其转化到大肠杆菌BL21(DE3)中,获得重组工程菌(E.coli(DE3/p28a/197)。该重组工程菌发酵的最佳接种量为5%～10%(v/v),且主要以包涵体形式表达,收获的菌体在高压均质机破碎压力为1 000 bar的条件下进行破碎,然后利用6 mol/L盐酸胍进行溶解变性,复性及经30 kD超滤膜包超滤后上纯化系统AKTA pure150 M,经一步阴离子交换层析进行纯化,其纯度可达98%以上,WB、分子量及圆二色谱等鉴定结果均与标准品一致。综上所述,成功建立大肠菌表达系统CRM197制备...     

抗HIV-1 gp120单链抗体导向SEAD227A融合蛋白原核温控型表达载体构建及表达

人工合成了能广泛识别HIV—1 gp120的单链抗体(SeFv120)基因和金黄色葡萄菌外毒素A(SEA)基因,将SEA基因第227位编码Asp(D)的密码子GAT转换成编码Ala(A)的密码子GCT,并对两基因进行密码子优化。构建原核温控型表达质粒pBV—120和pBV—SL120,经条件优化,pBV—120和pBV—SL120分别在E．coli BL21(DE3)plys中44℃诱导6h、42℃诱导7h获得最佳表达,表达量分别占菌体总蛋白的27．673％和32．519％。目的蛋白经包涵体洗脱、分子筛层析折叠复性后可与HIV—1抗原条发生良好的结合反应。     

Insertion of triangulated surfaces into a meccano tetrahedral discretization by means of mesh refinement and optimization procedures

In this paper, we present a new method for inserting several triangulated surfaces into an existing tetrahedral mesh generated by the meccano method. The result is a conformal mesh where each inserted surface is approximated by a set of faces of the final tetrahedral mesh. First, the tetrahedral mesh is refined around the inserted surfaces to capture their geometric features. Second, each immersed surface is approximated by a set of faces from the tetrahedral mesh. Third, following a novel approach, the nodes of the approximated surfaces are mapped to the corresponding immersed surface. Fourth, we untangle and smooth the mesh by optimizing a regularized shape distortion measure for tetrahedral elements in which we move all the nodes of the mesh, restricting the movement of the edge and surface nodes along the corresponding entity they belong to. The refining process allows approximating the immersed surface for any initial meccano tetrahedral mesh. Moreover, the proposed projection method avoids computational expensive geometric projections. Finally, the applied simultaneous untangling and smoothing process delivers a high‐quality mesh and ensures that the immersed surfaces are interpolated. Several examples are presented to assess the properties of the proposed method.     

Certification Institutions Expected to Update and Transfer Information on Present Standards

Standards are the basis for production enterprises to organize production, ex-factory inspection, trade (delivery) and technical exchanges, product certification, quality arbitration and supervision.……     

Flow calorimeter for enthalpy increment measurements on condensed gases

A flow calorimeter for enthalpy increment measurements on condensed gases is presented. A better knowledge of the properties of the liquefied natural gas is needed, and therefore a liquid loop has been designed for our flow calorimeter. The fluid loop in the calorimeter is designed in order to avoid the two-phase region, since two phases would give compositional disturbances in the measurements. The avoidance of the two-phase region is made possible by increasing the pressure of the test fluid after the measurement section, then heating the fluid at super-critical pressure past the critical point. Finally, the fluid is throttled to the low-pressure gas state at the inlet condition of the compressor that circulates the fluid. To perform the pressure increase, a new cryogenic pump has been designed. To evaluate the new equipment, measurements were taken on liquid ethane over the temperature range 146–256 K at pressure between 0.9 and 5.1 MPa.     

From Industry 4.0 to Research 4.0. Development of High Performance Fibers in the Light of Digitization

We associate a variety of innovations with the term ＂Industry 4.0＂. The pioneer of many 4.0 modifications forms the basisfor the trend towards the integrated di...     

Refrigerators Enter a New Era of Health Preservation Frestech initiates the establishment of China's first standard for household sterilization refrigerators

On November 30, 2007, the China Association for Standardization (CAS) held a press conference at Beijing Diaoyutai State Guest House. Leaders from the China Household Electric Appliance Research Institute, the China Household Electric Appliance Association, and the China Consumers' Association attended and made speeches.……     

Optimum-Simulation von Ernteprozessen

Zusammenfassung Bei der Gestaltung von Ernteprozessen — beispielsweise des Prozesses Mähdrusch, Körnertransport und -abnahme — als transportverbundene Fließarbeitsverfahren mit mindestens zwei verschiedenen Arbeitsmitteln gibt es einen großen Optimierungsspielraum. Solche Prozesse sind mehrstufige Bedienungsprozesse mit gemischter Anordnung der Bedienungseinrichtungen. Außerdem besitzen sie stark ausgeprägte räumliche Aspekte. Es wird vor allem gezeigt, welche Merkmale solcher Prozesse in Simulationsmodellen abgebildet sein müssen, welche Zielgrößen in Abhängigkeit von welchen Einflußgrößen unter Berücksichtigung welcher Prozeßbedingungen ermittelt werden sollten und wie der Bewertungsprozeß zur Ermittlung der gewünschten Informationen gestaltet werden muß.     

Roman iron and steel: A review

ABSTRACT

The production of ferrous metal increased during the Roman Late Republican period, Principate and Empire. The direct bloomery process was used to extract the metal from its ores using slag-tapping and slag-pit furnaces. The fuel was charcoal and an air blast was introduced by bellows-operated tuyères. Iron formed as a bloom, often as a spongy mass of metal, which contained impurities from the smelting process, including unreacted ore, fuel, slag and fragments from the furnace walls, while the metal was often inhomogeneous with varied carbon contents. Blooms were either smithed directly into bars or ingots or they were broken up, which also allowed the removal of gross impurities and a selection of pieces with similar properties to be made. These could then be forge-welded together and formed into characteristically shaped ingots. Making steel in the furnace seems to have been achieved: it depended on the ore and the furnace and conditions within it. Surface carburization was also carried out. Iron and steel were used extensively in construction and for tools and weapons. Fire welding was often used to add pieces of steel to make the edges of tools and weapons, which could be heat-treated by quenching to harden them.     

A study of centrifugal cast high boron high speed steel

In the present study a high‐boron high speed steel (HSS) roll material was designed. Many expensive alloy elements have been substituted by cheap boron alloy, and high‐boron high speed steel roll has been manufactured by centrifugal casting method. The microstructures, mechanical properties and wear resistance of centrifugal casting high‐boron high speed steel roll have been investigated by optical microscopy (OM), scanning electron microscopy (SEM), and X‐ray diffraction (XRD) analysis, hardness test, impact test and wear test. The results indicated that the solidification microstructures of high‐boron high speed steel roll consisted of M₂(B,C), (W,Mo)₂(B,C), M₃(B,C), M₂₃(B,C)₆ type borocarbides and martensite, a small amount of retained austenite. Borocarbides were continuously distributed over the grain boundary. After quenching from 1050 °C, local broken network appeared in partial borocarbides, and fine secondary borocarbide precipitated from the matrix. After tempering from 525 °C, the amount of precipitated borocarbide increased significantly. After heat treatment, the hardness of high‐boron high speed steel roll excelled 60 HRC, and its impact toughness excelled 8.0 J/cm². The single groove steel rolling amount of high‐boron high speed steel rolls increases by 500% than that of bainite cast iron roll, when the rolls are used in K₁ mill housing of bar mill.     

Thixotropy of Systems with Shear Thinning and Plastic Flow Behavior

The definition of the thixotropy is a decrease in viscosity with time in shear and a subsequent recovery of viscosity after the shear deformation is removed.We ...