含T细胞表位和B细胞表位的抗O型FMDV基因工程疫苗诱导豚鼠产生免疫反应

用FMDV疫苗免疫接种是预防口蹄疫的主要手段之一 .研制既含B细胞表位又含T细胞表位的基因工程疫苗对口蹄疫的防治具有更强的保护能力 .用化学方法合成O型口蹄疫病毒 (FMDV)外壳蛋白VP1中 2 1～ 40位肽段的T细胞表位基因 ,与 1 41～ 1 60肽段的B细胞表位基因串联后 ,与 β 半乳糖苷酶基因相连 ,构建成一重组质粒 ,并在大肠杆菌中表达出融合蛋白疫苗 .动物实验表明 ,该疫苗不仅能诱导豚鼠产生滴度很高的中和抗体 ,而且产生大量对病毒专一性T细胞 ,与不含 2 1～ 40肽段的疫苗相比 ,用该疫苗免疫的豚鼠血单个核细胞对FMDV的反应性提高 7倍以上 ,并能抵抗病毒攻击 .说明该疫苗可同时激活体液免疫及细胞免疫反应 ,有较强的保护作用 .     

MS2介导的口蹄疫类病毒颗粒疫苗的研究

研究表明,口蹄疫病毒(Foot-and-mouth disease virus,FMDV)的VP1蛋白含有关键的抗原决定簇.根据O/HL-JOC12/03猪源FMDV毒株VP1上第141～160位氨基酸序列设计引物,利用重叠延伸PCR(SOE PCR)技术将该序列插入在大肠杆菌(Escherichia coli)噬菌体MS2外壳蛋白基因的特定位点.然后连接到原核表达载体pET28a上,构建了重组表达质粒28a-CP-VP1,转化BL21(DE3),ITPG诱导表达得到融合表达的CP-VP1.透射电子显微镜下观察融合蛋白CP-VP1成类病毒颗粒(virus-like particles,VLPs)状.间接ELISA实验证实该蛋白能够特异地结合豚鼠抗O型FM-DV的阳性血清,30μg的CP-VP1的VLPs蛋白免疫小鼠后可以诱导其产生高效价的特异抗体.故该口蹄疫病毒抗原决定簇展示在噬菌体MS2上表面的VLPs蛋白具有开发成口蹄疫疫苗的前景.     

A型口蹄疫病毒VP1免疫活性肽基因串联片段的化学合成及克隆与表达

A型口蹄疫病毒（FMDV）VP1蛋白的141～160和200～213位氨基酸序列是决定FMDV免疫原性的抗原决定簇片断．人工合成一个免疫活性肽基因的串连片段（20AA－14AA-20AA）、全部选用大肠杆菌偏爱密码子．在5’端带有EcoR1位点和一个起始密码子ATG，在3’端带有BamH1位点和一个终止密码子TGA．利用这两个酶切位点把该基因连接到pWR590质粒上．并筛选高表达的菌株．结果表明该融合蛋白在大肠杆菌JM101中能高表达．经斑点ELISA实验证明．大肠杆菌细胞中表达的融合蛋白有A型FMDV抗原活性．     

AsiaⅠ型口蹄疫病毒基因工程多肽疫苗的构建及其免疫原性分析

为了构建Asia Ⅰ型口蹄疫病毒多肽疫苗,用反转录PCR方法扩增VPI基因并测得该基因的核苷酸序列．将VPI（133AA-163AA）、VP2（1AA～33AA）抗原表位基因分别与卢一半乳糖苷酶基因（简称LacZ＇）3’末端相连构建表达质粒LacZ’-VIP和LacZ’-VP2．化学合成VP1（133AA～163AA）-VP2（1AA～33AA）-VPl（133AA～163AA）串联重复抗原表位DNA,然后分别连接在β-半乳糖苷酶基因和IgG重链恒定区基因的3’末端,构建两种重组表达质粒LacZ’-VPl（133AA--163AA）-VP）2（1AA～33AA）-VP1（133AA-163AA）（命名为pTl）和IgGvPl（133AA～163AA）-VP2（1AA～33AA）-VPl（133AA-163AA）（命名为pT2）．通过Westernblot、血清中和抗体效价测定、T细胞增殖反应及豚鼠抗病毒保护实验分析所构建疫苗的免疫原性及有效性．结果显示pT1、pT2表达的融合蛋白能够诱发豚鼠产生较高的中和抗体及刺激豚鼠T细胞增殖．pT1、pT2两种融合蛋白分别2次免疫豚鼠后,100％的豚鼠能抵抗100 ID50 AsiaⅠ型口蹄疫病毒攻击．pT2融合蛋白1次免疫豚鼠后70％的豚鼠能抵抗100 ID50 AsiaⅠ型口蹄疫病毒攻击．     

O型口蹄疫病毒VP4蛋白一个保守性表位的精细鉴定

口蹄疫病毒(FMDV)的结构蛋白VP4在7个血清型之间是非常保守的,对其抗原表位最小基序的测定有助于开发广谱性多肽疫苗.本研究采用生物合成肽法,利用豚鼠抗FMDV O型标准血清,对最新流行的FMDV O型毒株O/BY/CHA/2010的VP4蛋白进行了线性表位扫描作图研究.结果表明:在覆盖VP4蛋白全长序列的10个18聚肽中,发现1个能与豚鼠抗FMDV O型标准血清发生免疫印迹反应的抗原性肽VP4-3;B细胞表位鉴定确定了抗体识别VP4-3的最小基序是INNYYM;该表位位于VP4蛋白的三维结构表面;对7个FMDV血清型的122个毒株同源蛋白质进行序列比对,发现这一表位在7个FMDV血清型的120个毒株中是100%保守.     

O型口蹄疫病毒VP1基因重组山羊痘病毒活载体疫苗的研究

旨在构建能够表达O型口蹄疫病毒(FMDV)VP1基因的重组山羊痘病毒(GPV),并以此为活载体疫苗,评估其免疫效力.将FMDV的VP1基因插入转移质粒pTKfpgigp中,构建重组转移质粒pTKfpgigp-VP1,并转染已感染GPV的羔羊睾丸(LT)细胞,产生重组GPV(rGPV),筛选纯化rGPV,检测rGPV在LT细胞中VP1基因的表达情况.将获得的rGPV皮内接种山羊,检测抗山羊痘(GP)和口蹄疫(FMD)特异性抗体水平.结果表明,获得含有FMDV的VP1基因重组毒株rGPV/FMDVVP1,在LT细胞中VP1基因进行正常转录,其表达产物能与FMDV抗血清产生特异性反应.rGPV能诱导产生不同水平的抗GP和FMD的特异性抗体.可为FMD重组标记疫苗的研制提供参考.     

含FMDV中和表位的重组病毒颗粒的构建及鉴定

针对FMDV的疫苗研究一直是防控口蹄疫的重点,而病毒样颗粒(VLPs)形式的疫苗因其安全性和近似传统灭活疫苗的良好免疫效果,近年来逐渐成为疫苗研究的一个新方向.本实验利用猪圆环病毒PCV2的衣壳蛋白(CAP)作为载体,分别插入FMDV MYA98毒株的抗原表位组合——VP1蛋白上B细胞表位141~160aa(B)与串联的B细胞和T细胞表位141~160aa-21~40aa-141~160aa(BTB),构建对应重组杆状病毒,分别命名为reBAC-CAP-B,reBAC-CAP-BTB.将上述病毒颗粒转入Sf9细胞表达,收获重组杆状病毒并再次感染Sf9细胞扩大病毒滴度,获得目的蛋白CAP-B,CAP-BTB,经过SDS-PAGE,Western blot鉴定正确,透射电镜观察到20~30nm大小的目的颗粒.     

含T细胞表位和B细胞表位的抗O型FMDV基因工程疫苗诱导豚 …

用ＦＭＤＶ疫苗免疫接种是预防口蹄疫的主要手段之一,研制既含Ｂ细胞表位又含Ｔ细胞表位的基因工程疫苗对口蹄疫的防治具有更强的保护能力,用化学方法合成Ｏ型口蹄疫病毒（ＦＭＤＶ）外壳蛋白ＶＰ１中２１～４０位肽段的Ｔ细胞表位基因,与１４１～１６０肽段的Ｂ细胞表位基因串联后,与β－半乳糖苷酶基因相边,构建成一重组质粒,并在大肠杆菌中表达出融合蛋白疫苗,动物实验表明,该疫苗不仅能诱导豚鼠产生滴度很高的中和抗体,     

家畜口蹄疫新型疫苗研制与生产工艺创新2014年度报告

<正>在研制的口蹄疫新型疫苗有10多种,分别是纳米微球黏膜免疫疫苗,表位肽疫苗、口蹄疫O型复合表位蛋白疫苗、A型重组毒株(Re-A/WH/2009)的口蹄疫O型、A型、亚洲Ⅰ型三价灭活疫苗、猪口蹄疫O型广谱基因工程病毒灭活疫苗、口蹄疫O型标记疫苗、猪O型Mya98毒株空衣壳疫苗、猪口蹄疫O型合成肽疫苗(多肽2600+2700+2800)、牛口蹄疫(O型、Asia1型)二价合成肽疫苗、以及口蹄疫病毒活载体疫苗。其中取得突破性进展的有5种,牛口蹄疫(O型、Asia1型)二价合成肽疫苗PD50值大于6.0,免疫持续期为6个月,保存期为12个月,已完成所有实验室研究和临床试验,申请了新兽药注册并已通过初审;猪口蹄疫O型合成肽疫苗(多肽2600+2700+2800)PD50值大于6.0,免疫持续期为6个月,保存期为12个月,已完成所有实验室研究和临床试验,现已申请新兽药注册并通过复核试验和复审;含A型重组毒株(Re-A/WH/2009)的口蹄疫O型、A型、亚洲Ⅰ型三价灭活疫苗PD50值大于6.0,免疫持续期为6个月,保存期为12个月,获得了农业部新兽药注册证书,实现了产业化;猪口蹄疫O型广谱基因工程病毒灭活疫苗已申报农业部临床试验批文;口蹄疫O型标记疫苗已完成疫苗质量研究,并已申请新兽药注册。其他疫苗研究也均取得了程度不等的进展。利用反向遗传操作技术平台,获得基因工程修饰病毒疫苗株5株:(1)r V-STN-5;(2)9O/r V-1;(3)Re-A/WH/2009;(4)Re-Mya/98/BY/2010;(5)Re-Asia1/HN/2006。利用基因克隆、重组等技术,获得其他基因工程修饰病毒9株,分别为:(1)表达O型FMDV不同亚型主要免疫原性基因的重组伪狂犬病毒1株;(2)共表达O-A-Asia1型FMDV主要免疫原性基因的重组伪狂犬病毒1株;(3)表达FMDV免疫原性基因的杆状病毒2株;(4)表达猪源IFN-α/IFN-γ和A型FMDV P1基因的重组杆状病毒3株;(5)表达口蹄疫和小反刍兽疫病毒主要保护性抗原基因的重组羊痘病毒2株。成功研制Cp G-IFN、Cp G-IL4以及多磷腈和Cp G DNA等生物复合佐剂3种,纳米乳油佐剂、纳米粒IL-2佐剂以及多孔硅和上转换荧光纳米材料的载药系统等纳米复合佐剂材料4种,以及IL-2、IL-4和IFN-γ等多种哺乳动物细胞表达质粒和多糖类免疫增强剂4种。     

多个发夹RNA的混合使用抑制O型和AsiaI型口蹄疫病毒的复制

通过对国内流行的口蹄疫病毒（FMDV）毒株的序列分析,锁定RNA干扰的3个目标基因即3D,VP4和2B,分别编码聚合酶POL,病毒结构蛋白VP4和非结构蛋白2B,这3个靶基因序列在不同流行毒株之间相对保守．分别设计合成靶向这些基因序列的发夹RNA（short-hairpin RNA,shRNA）表达模板,构建了5个shRNA表达质粒p3D-NT56,p3D-NT19,pVP4-NT65,pVP4-NT19和p213-NT25．进行单独应用或混合应用,于细胞水平和乳鼠水平检测其抗病毒活性．结果显示,3D-NT56／19,VP4-NT65／19和2B—NT25 shRNA对O型FMDV均有抑制作用,但是对Asia I型FMDV,仅2B-NT25 shRNA具有较显著的抑制作用．3个shRNA表达质粒的混合使用（p3D-NT56＋pVP4-NT65＋p2B-NT25,p3D-NT19＋pVP4-NT19＋p213-NT25）,不仅能够交叉抑制不同血清型FMDV的复制,而且这种抑制作用较单一shRNA延续更长的时间．     

Characterization of Ordovician carbonate reservoirs, southeastern Saskatchewan, Canada

The discovery of the prolific Ordovician Red River reservoirs in 1995 in southeastern Saskatchewan was the catalyst for extensive exploration activity which resulted in the discovery of more than 15 new Red River pools. The best yields of Red River production to date have been from dolomite reservoirs. Understanding the processes of dolomitization is, therefore, crucial for the prediction of the connectivity, spatial distribution and heterogeneity of dolomite reservoirs.The Red River reservoirs in the Midale area consist of 3～4 thin dolomitized zones, with a total thickness of about 20 m, which occur at the top of the Yeoman Formation. Two types of replacement dolomite were recognized in the Red River reservoir: dolomitized burrow infills and dolomitized host matrix. The spatial distribution of dolomite suggests that burrowing organisms played an important role in facilitating the fluid flow in the backfilled sediments. This resulted in penecontemporaneous dolomitization of burrow infills by normal seawater. The dolomite in the host matrix is interpreted as having occurred at shallow burial by evaporitic seawater during precipitation of Lake Almar anhydrite that immediately overlies the Yeoman Formation. However, the low δ18O values of dolomited burrow infills (-5.9‰～ -7.8‰, PDB) and matrix dolomites (-6.6‰～ -8.1‰, avg. -7.4‰ PDB) compared to the estimated values for the late Ordovician marine dolomite could be attributed to modification and alteration of dolomite at higher temperatures during deeper burial, which could also be responsible for its 87Sr/86Sr ratios (0.7084～0.7088) that are higher than suggested for the late Ordovician seawaters (0.7078～0.7080). The trace amounts of saddle dolomite cement in the Red River carbonates are probably related to "cannibalization" of earlier replacement dolomite during the chemical compaction.     

A computer generator for randomly layered structures

AcomputergeneratorforrandomlylayeredstructuresYUJia shun1,2,HEZhen hua2(1.TheInstituteofGeologicalandNuclearSciences,NewZealand;2.StateKeyLaboratoryofOilandGasReservoirGeologyandExploitation,ChengduUniversityofTechnology,China)Abstract:Analgorithmisintrod…     

海南岛地体及其毗邻陆缘晚中生代—新生代古地磁研究和构造演化

本文叙述了对海南岛及其毗邻大陆边缘白垩纪到第四纪地层岩石进行古地磁研究的全部工作过程。通过分析岩石中剩余磁矢量的磁偏角及磁倾角的变化,提出海南岛白垩纪以来经历的构造演化模式如下:早期伴随顺时针旋转而向南迁移,后期伴随逆时针转动并向北运移。联系该地区及邻区的地质、地球物理资料,对海南岛上述的构造地体运动提出以下认识:北部湾内早期有一拉张作用,主要是该作用使湾内地壳显著伸长减薄,形成北部湾盆地。从而导致了海南岛的早期构造运动,而海南岛后期的构造运动则主要是受南海海底扩张的影响。海南地体运动规律的阐明对于了解北部湾油气盆地的形成演化有重要的理论和实际意义。     

Exciton Seebeck in Molecular Systems

Various applications relevant to the exciton dynamics,such as the organic solar cell,the large-area organic light-emitting diodes and the thermoelectricity,are operating under temperature gradient.The potential abnormal behavior of the exicton dynamics driven by the temperature difference may affect the efficiency and performance of the corresponding devices.In the above situations,the exciton dynamics under temperature difference is mixed with     

Achieving Unusual States of Matter Under High Pressure

The elongation method,originally proposed by Imamura was further developed for many years in our group.As a method towards O(N)with high efficiency and high accuracy for any dimensional systems.This treatment designed for one-dimensional(ID)polymers is now available for three-dimensional(3D)systems,but geometry optimization is now possible only for 1D-systems.As an approach toward post-Hartree-Fock,it was also extended to     

《Chinese Science Bulletin》SUBJECT INDEX TO VOLUME 59(2014)

正~~     

An on-line scaling method for improving scalability of a database cluster

The explosive growth of the Internet and database applications has driven database to be more scalable and available, and able to support on-line scaling without interrupting service. To support more client's queries without downtime and degrading the response time, more nodes have to be scaled up while the database is running. This paper presents the overview of scalable and available database that satisfies the above characteristics. And we propose a novel on-line scaling method. Our method improves the existing on-line scaling method for fast response time and higher throughputs. Our proposed method reduces unnecessary network use, i.e. , we decrease the number of data copy by reusing the backup data. Also, our on-line scaling operation can be processed parallel by selecting adequate nodes as new node. Our performance study shows that our method results in significant reduction in data copy time.     

An improved R-tree based on childnode''s probability

R-Tree is a good structure for spatial searching. But in this indexing structure,either the sequence of nodes in the same level or sequence of traveling these nodes when queries are made is random. Since the possibility that the object appears in different MBR which have the same parents node is different, if we make the subnode who has the most possibility be traveled first, the time cost will be decreased in most of the cases. In some case, the possibility of a point belong to a rectangle will shows direct proportion with the size of the rectangle. But this conclusion is based on an assumption that the objects are symmetrically distributing in the area and this assumption is not always coming into existence. Now we found a more direct parameter to scale the possibility and made a little change on the structure of R-tree, to increase the possibility of founding the satisfying answer in the front sub trees. We names this structure probability based arranged R-tree (PBAR-tree).     

Integrating GIS Web services based on mediating architecture

The geographic information service is enabled by the advancements in general Web service technology and the focused efforts of the OGC in defining XML-based Web GIS service. Based on these models, this paper addresses the issue of services chaining,the process of combining or pipelining results from several interoperable GIS Web Services to create a customized solution. This paper presents a mediated chaining architecture in which a specific service takes responsibility for performing the process that describes a service chain. We designed the Spatial Information Process Language (SIPL) for dynamic modeling and describing the service chain, also a prototype of the Spatial Information Process Execution Engine (SIPEE) is implemented for executing processes written in SIPL. Discussion of measures to improve the functionality and performance of such system will be included.     

Tracking data management using topology of moving object database system for moving object constrained trajectory in LBS application

Advances in wireless technologies and positioning technologies and spread of wireless devices, an interest in LBS (Location Based Service) is arising. To provide location based service, tracking data should have been stored in moving object database management system (called MODBMS) with proper policies and managed efficiently. So the methods which acquire the location information at regular time intervals then, store and manage have been studied. In this paper, we suggest tracking data management techniques using topology that is corresponding to the moving path of moving object. In our techniques, we update the MODBMS when moving object arrived at a street intersection or a curved road which is represented as the node in topology and predict the location at past and future with attribute of topology and linear function. In this technique, location data that are corresponding to the node in topology are stored, thus reduce the number of update and amount of data. Also in case predicting the location,because topology are used as well as existing location information, accuracy for prediction is increased than applying linear function or spline function.