牙鲆生长激素基因的克隆及其在大肠杆菌中的融合表达

采用逆转录聚合酶链式反应(RT-PCR)方法,从牙鲆(Paralichthys olivaceus)脑垂体总RNA中扩增出编码牙鲆生长素(GH)成熟肽基因序列。重组至融合表达载体pGEX-4T-3中,构建成牙鲆GH基因融合表达载体pGEX-gh,转化大肠杆菌BL21(DF3),筛选阳性克隆,IVIG诱导表达。经SDS-PAGE电泳显示在45kD处有特异的蛋白条带出现,该蛋白的表达量随诱导时间的延长而增加,3h达最高值,达到细胞总蛋白的18．3％。该融合蛋白在胞内主要以包涵体状态存在,经优化表达条件,成功地获得了可溶性的融合蛋白,经Glutathione Sepharase 4B凝胶纯化后用Western-blotting检测表明其为牙鲆生长激素,并通过酶联免疫吸附受体法证实其具有生物学活性。     

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 )。     

斑马鱼胸腺苷酸合成酶基因的克隆与表达

用RT-PCR和RACE方法,从斑马鱼胚胎中克隆出胸腺苷酸合成酶(TS)的cDNA全长序列,分析表明,它的编码框序列含有954个核苷酸,编码一个分子量为36kD的318个氨基酸的蛋白序列.将斑马鱼TS的cDNA序列连接到pET-28表达载体上,构建了一个表达载体pET-28/Z-TS,并在大肠杆菌BL21(DE3)中进行了IPTG诱导表达,纯化的TS蛋白在28℃表现出比较高的生物活性,与人的TS蛋白的Km值比较接近.     

用弱化dhfr双顺反子载体在CHO细胞中高效表达低分子量尿激酶突变体

将二氢叶酸还原酶基因(dhfr)克隆至pIRES载体中弱化的脑心肌炎病毒(ECMV)内部核糖体进入位点(IRES)的下游,构建了含有弱化dhfr筛选标记和可用氨甲蝶呤(MTX)加压提高表达水平的双顺反子真核表达载体pIRES-dhfr.利用该表达载体表达了一种无糖基化和具有凝血酶抗性的低分子量尿激酶型纤溶酶原激活剂(LMW-uPA)突变体(缺失尿激酶原的1-143位氨基酸,Arg156→Lys,Asn302→Ala),用脂质体转染方法将表达载体pIRES-dhfr/LMW-UK转染CHO-dhfr-细胞后经过一轮MTX筛选,几乎所有获得的单克隆细胞株都表达LMW-UK,其中约50%为表达水平较接近(500-5000 IU/106cells/d)的高表达阳性克隆.用转瓶无血清培养表达水平约为17.5pg/cell/d的rCHO细胞系LB2-UK,收集的上清经过阳离子交换柱和凝胶过滤层析两步纯化后,获得的重组蛋白的纯度可以达到99%.用S-2444发色底物法检测,所获得的突变体双链UK比例大于98%.     

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.     

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。     

苏丹红Ⅰ号多克隆抗体的制备与特异性鉴定

采用琥珀酸酐法,分别以BSA、OVA为载体蛋白合成了免疫抗原和包被抗原并通过SDS-PAGE凝胶电泳和紫外扫描法鉴定了合成的抗原为载体蛋白和苏丹红I号的复合物.将合成的免疫抗原用弗氏佐剂乳化后免疫新西兰大白兔,制备出多克隆抗体,经辛酸纯化,间接ELISA法测得有高效价血清抗体;进一步采用琼脂双扩散试验和免疫金试纸法鉴定出血清中含有苏丹红Ⅰ号特异性抗体.     

牙鲆抗菌肽hepcidin基因在大肠杆菌中的融合表达

根据从实验室克隆的牙鲆抗菌肽hepcidin基因(GenBank登陆号DQ129693)的序列,设计了特异性引物,PCR扩增其成熟肽片段.重组至融合表达载体pGEX-4T-1中,构建成牙鲆抗菌肽hepcidin基因融合表达载体pGEX-fhep,转化至大肠杆菌BL21(DE3)plyS中,筛选得到的阳性克隆用IPTG进行诱导.SDS-PAGE电泳显示在29kD处有特异性的蛋白条带出现,Western-blotting 检测表明已经成功表达了融合蛋白,表达的融合蛋白最高占总蛋白的21%.纯化得到的GST-fhep用凝血酶切割后,用亲合层析得到牙鲆的抗菌肽hepcidin.抑菌实验结果表明抗菌肽对大肠杆菌(ATCC25922)有一定程度的抑制作用.     

壳聚糖-激素药条埋植技术初探

采用壳聚糖-激素共沉淀方法,制备了新型的壳聚糖-甲基睾酮（chitosan-MT）激素埋植药条。药条埋植于实验鱼8天和15天后分别取血样检测GtH含量和解剖实验鱼观察药条的缓释情况,采用放射免疫检法检测血清GtH含量,结果表明,chitosan-MT埋植8天和15天后雄鱼的血清GtH平均含量分别提高到原来的4．36倍和4．53倍;而雌鱼的GtH含量分别是实验前的2．76倍和2．74倍;但同时期取样的雌、雄对照组鲤鱼血清GtH平均含量没有明显差异。由此可见,制备的chitosan-MT药条能在鲤鱼机体内稳定而缓慢释放,并能通过正反馈途径促进GnRH的释放,从而使血清GtH含量增加。     

中国明对虾线粒体MnSOD在大肠杆菌中的重组表达、产物纯化及活性测定

采用PCR方法扩增编码中国明对虾线粒体锰超氧化物歧化酶(MnSOD)成熟肽的基因,并克隆到大肠杆菌表达载体pCRR T7/NT TOPOR TA中进行体外重组表达.重组质粒转化大肠杆菌BL21 (DE3) pLysS后,经IPTG诱导表达产生包涵体形式的目的蛋白.对重组蛋白进行 LC-ESI-MS 分析的结果表明,融合蛋白的三个肽段与中国明对虾线粒体MnSOD 相应肽段完全一致.将重组蛋白通过金属螯合柱进行纯化,进而透析、复性,最后获得了活性高达2373U/mg的重组蛋白.中国明对虾线粒体MnSOD的成功表达,为深入研究其在中国明对虾免疫反应和抗氧化胁迫中的作用奠定了基础.     

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.     

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...     

The Research of Anti-Wear Properties of Organomolybdenum Compounds in the Engine Oils

A four-ball tester was used to evaluate the anti-wear performance of three kinds of organomolybdemun compounds in the engine oils, i. e., molybdenum dialkyldithiophosphate (MoDDP), molybdenum dialkyldithiocarbamate ( MoDTC), and sulphur and phosphorus freeorganomolybdeum (Molybdate). The results indicate that a low concentration of MoDDP doesn' t improve the anti-wear properties of the commercial engine oils, but a high concentration of MoDDP can obviously improve the anti-wear properties and the load-carrying capacity of the engine oils. MoDTC doesn' t improve the antiwear properties of the engine oils, but worsens the anti-wear properties of the oils. Signifi can timprove ment of frictional and wear characteristics is obtained with Molybdate added in the commercial engine oils and the formulated oils.     

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.     

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.     

Synthesis of Alumina-Zirconia Nanocomposites by Solgel Process

Al₂O₃-ZrO₂ nanocomposites were developed starting with the solgel process. Composite alumina-zirconia nanopowders were synthesized from metallorganic precursors (Aluminium secondary butoxide and zirconium Iso propoxide) using the solgel process. The parameters affecting the synthesis—solvent, concentration of precursor, R/H ratio (i.e., dilution of water in solvent)—were varied as also the temperature and pH. BET and TEM were used to measure nanosize. Diffuse reflectance spectroscopy and also qualitative optical absorption led to identical particle size estimate. The variation of process parameters was used to study the effect and interdependence of process parameters. Artificial Neural Networks was used to rigorously analyze the process. Although this led to confirmation of interdependence of parameters, the presence of a single overwhelming solvent variable was also established. Then the optimal process was used to synthesize more nanopowder. To produce bulk nanocomposite the nanopowders were sintered by varying the temperature and time period. The sintered lithoids were probed with a vickers hardness tester to measure elastic modulus, hardness, and fracture toughness. The results showed high elastic modulus, modest hardness, and very high fracture toughness.     

Certification Becomes Important for the I Quality; and Safety Supervision of the Chinese Liquor Sector

The end of 2007, over 200 unit products of more than 80 Chinese firms have passed the quality grade certification for liquor products. These products involve distilled spirits, beer, wine, yellow wine, fruit syrup wine and others, and cover over 80% of the national top-branded liquor products.……     

Deposition and Coating Properties on CVD Tungsten

Surface characterization and microstructure studies are performed on chemical vapor deposited (CVD) tungsten coating. There is about 2 μm thickness diffusion layer of tungsten in the molybdenum substrate. The thermal shock test shows tungsten coating has good adhesion with molybdenum substrate, but the elements of oxygen and carbon in the tungsten coating have the bad affection to the adhesion. The result of high-temperature diffusion experiment is the diffusion rate from molybdenum substrate to tungsten coating is faster.