中国明对虾蜕皮抑制激素在大肠杆菌中的表达与分离纯化

采用大肠杆菌表达系统对中国明对虾蜕皮抑制激素(MIH)进行了体外重组表达研究.重组蛋白以包涵体的形式存在于菌体中.尿素-SDS-PAGE分析表明,经1mmol/L的IPTG诱导4h后,重组蛋白获得了大量表达,在分子量为13 kD处有一条与预测大小一致的特异性蛋白条带;经金属螯合柱纯化后,得到了电泳纯的融合蛋白.Western blotting检测结果表明:重组表达的融合蛋白与兔抗刀额新对虾MIH的多克隆抗体特异结合,证实该融合蛋白为中国明对虾MIH.通过胶内酶切与LC-ESI-MS分析进一步证实融合蛋白的部分肽段与日本对虾MIH一致.MIH融合蛋白的成功表达为进一步深入研究其在中国明对虾蜕皮过程分子作用机制奠定了基础.     

中国明对虾凝血栓蛋白基因全长cDNA的克隆及其表达特征

获得了中国明对虾凝血栓蛋白(TSP)基因全长cDNA,该基因由2868个碱基组成,具有一个2763bp的开放阅读框,编码920个氨基酸,其中包含21个氨基酸组成的信号肽.中国明对虾TSP由四类不同的结构域组成,从N端开始顺序为几丁质结合结构域,EGF-like结构域,Type Ⅲ repeat和TSP carboxyl-terminal domain(TSP-C结构域).结构域分析发现,中国明对虾的N端不具有TSP N-terminal domain(TSPN结构域),但有一个几丁质结合结构域,其C端的Type Ⅲ和TSP-C结构域非常保守.该基因在弧菌感染后的对虾淋巴器官和肝胰脏中的表达量显著增加,并具有不同的时空表达趋势,提示中国明对虾TSP基因在免疫反应中具有重要作用.     

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.     

WSSV感染中国对虾肌肉与鳃膜蛋白变化及差异蛋白的鉴定

采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、基质辅助激光解吸电离飞行时间质谱分析(MALDI-TOF/ MS)、生物信息学技术,研究了WSSV感染中国对虾后鳃膜蛋白和肌肉组织蛋白的变化.结果表明,WSSV感染中国对虾12h后,鳃膜蛋白27kD蛋白增强,至72h, 27kD蛋白增强显著.肌肉组织蛋白在感染48h后,出现1 条明显差异分子量为22kD的蛋白带.通过对该差异蛋白进行MALDI -TOF/ MS的肽质量指纹图谱及其生物信息分析,鉴定为钙离子结合蛋白,其在对虾抗病毒感染免疫中的功能值得关注.     

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。     

华根霉(Rhizopus chinensis)前导肽脂肪酶基因的克隆及其在Pichia pastoris中的表达

通过3次PCR程序克隆得到了华根霉(Rhizopus chinensis CCTCCM201021)脂肪酶全基因序列,该基因的开放阅读框长1170bp,不含内含子,编码一个389个氨基酸残基的蛋白质,包括26个氨基酸的信号肽,94个氨基酸的前导序列和269个氨基酸的成熟肽,其推断的氨基酸序列与一些已报道的根霉脂肪酶序列同源性为86%.在巴斯德毕赤酵母(Pichia pastoris)GS115中分泌表达前导肽序列.SDS-PAGE分析表明表达蛋白的分子量约37kD.N-端氨基酸序列分析表明该重组蛋白分泌过程中前导序列N-端的67个氨基酸被切割掉,表达的重组脂肪酶由前导序列C-端的27个氨基酸和成熟肽的269个氨基酸组成.发酵132h后上清中重组脂肪酶的表达量最高,蛋白含量约5.4mg/mL,橄榄油乳化法测水解酶活为161U/mL,其比活比野生型华根霉脂肪酶高约43倍.     

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。     

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...     

毛细管电泳RT-PCR方法在中国明对虾抗菌肽基因表达定量检测中的应用

建立了毛细管电泳RT—PCR检测基因表达谱的方法,并利用实时定量PCR技术对此方法进行基因表达定量的可靠性进行了验证。利用此新方法研究了弧菌和白斑综合征病毒（WSSV）感染对中国明对虾抗菌肽（penaeidin）基因mRNA表达的影响。结果表明,弧菌感染后对虾肽基因表达变化可分为三个阶段：迅速下调,缓慢回升,持续稳定。WSSV感染后对虾肽基因表达的变化显示出与弧菌感染组不同的变化趋势：迅速下调,迅速回升,再次下降。     

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.     

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.     

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.     

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.……     

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.     

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.     

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.