鸭疫里氏杆菌外膜微孔蛋白基因的克隆和序列分析及PCR检测方法的建立

根据GenBank中的鸭疫里氏杆菌(RA)外膜磷酸盐选择性微孔蛋白(Porin)基因序列设计特异性引物,以血清1、2、6、10、11、13、14和17型RA菌株的基因组DNA为模板均扩增出大小为1 212bp的目的片段,序列比对显示该基因在8种血清型菌株间同源性均大于99.6%。针对保守性最高的357bp片段设计特异性引物,建立了PCR检测方法,对鸭致病性大肠杆菌、鼠伤寒沙门菌、鸭巴氏杆菌及鸭肝炎病毒均未扩增出目的条带,表明该方法具有较高的特异性;敏感性试验显示最小检出量为73.96pg的基因组。应用建立的PCR方法对分离自吉林、河北及北京的8个菌株进行扩增,均在357bp处扩增出清晰的目的条带,与细菌分离鉴定的符合率达100%。本研究为RA的鉴定提供了新方法,为RA外膜微孔蛋白的后续研究提供了理论依据。     

沙门菌CRISPR与耐药性的关系及生物信息学分析

为研究沙门菌成簇间隔短回文重复序列(CRISPR),对CRISPR进行生物信息学分析,为进一步研究CRISPR功能及其对细菌耐药的影响奠定基础。对合肥市宠物医院分离鉴定的14株沙门菌进行耐药性(24种抗菌药物)和CRISPR检测,分析CRISPR与沙门菌多重耐药的关系。对沙门菌PCR产物进行测序,通过BLAST、RN A二级结构预测、CRISPR Target等生物信息学方法对沙门菌的CRISPR进行生物信息学分析。结果显示,沙门菌均对3种及3种以上药物耐药,有8株菌检测到CRISPR 1,14株菌检测到CRISPR2;CRISPR阳性菌株均为多重耐药菌株,CRISPR序列差异性与耐药性之间没有直接相关性,CRISPR序列与血清型之间有一定相关性。8株CRISPR 1阳性沙门菌重复序列均含1条(9、13号除外)长度为29 bp的重复序列,分为4类,14株沙门菌CRISPR 2的重复序列共有18类,其RNA二级结构都能形成回文结构,但茎环大小有差异。同源性比对发现,CRISPR 1中81.6%的间隔序列与质粒的基因组序列具有同源性,18.4%与噬菌体有同源性;CRISPR 2中80%的间隔序列与质粒具有同源性,16.8%与噬菌体有同源性,3.2%与细菌有同源性。结果表明,携带CRISPR的沙门菌广泛存在,CRISPR可能与沙门菌血清型相关,明确了CRISPR结构的生物学信息。     

猪源嗜麦芽窄食单胞菌16 S rRNA基因的克隆和序列分析

从临床病猪无菌采集抗凝血 ,抽提全血基因组DNA ,用能够扩增大多数真细菌 16SrRNA基因的通用引物 ,扩增出长度为 15 0 2bp的嗜麦芽窄食单胞菌的 16SrRNA基因 ;该基因与国外报道的嗜麦芽窄食单胞菌部分临床和环境分离株核苷酸序列的同源性达 99%以上。证实嗜麦芽窄食单胞菌可感染猪。     

应用复合PCR方法检测沙门菌的研究

参照文献报道的沙门菌Repeatse和hisJ基因片段的引物序列 ,设计并合成了 2对引物 ,对沙门菌属和非沙门菌属的标准菌株及分离菌株进行了复合PCR扩增反应。结果 ,沙门菌PCR产物均出现 199bp与 4 95bp的特异性DNA扩增条带 ,而非沙门菌均未出现扩增条带 ,证明这 2对引物具有沙门菌属特异性。将提取的沙门菌DNA做梯度稀释 ,测定该复合PCR体系的敏感度。结果表明 ,此体系可检出 10 3CFU/mL菌液提取的DNA模板。应用上述方法 ,对进境鱼粉阳性样品进行了检测 ,均出现阳性结果。本研究表明 ,复合PCR是一种特异、敏感、快速的沙门菌检测方法 ,可应用于口岸系统鱼粉、肉骨粉的日常检测。     

牛化脓隐秘杆菌的分离及其主要毒力基因的PCR鉴定

从2009-2012年多个奶牛场送检的5份以奶牛化脓性炎症为特征的病料中分离出5株细菌,根据其革兰氏染色的形态特征、培养特性、生化特性以及16SrRNA基因序列的BLAST分析,确定分离菌为化脓隐秘杆菌(Trueperella pyogenes),并对分离菌的主要毒力因子基因进行了PCR鉴定和动物试验。结果显示,5株分离菌的16SrRNA基因序列与GenBank中收录的11株牛源化脓隐秘杆菌16SrRNA基因序列的同源性为99%以上。HLJ-1005株分离菌基因组中缺失胶原结合蛋白(CbpA)基因,但包含溶血素(PLO)基因、神经氨酸酶H(NanH)基因和神经氨酸酶P(NanP)基因,且HLJ-1005株分离菌的致病力明显弱于其他4株分离菌。证实,化脓隐秘杆菌的致病力与cbpA基因和nanP基因的存在相关。     

致病性沙门菌SYBR GreenⅠ实时荧光定量PCR检测方法的建立

为建立一种灵敏、特异、快速地检测食品中致病性沙门菌的方法,针对沙门菌致病性菌株特有的invA基因设计引物,PCR扩增目的片段,构建重组质粒pMD19-T-inv285,将其作为标准品进行real-time PCR反应体系的优化和标准曲线的绘制,并进行灵敏度、特异性及稳定性检测;同时用该方法对90份肉品样品进行检测。结果显示,建立的致病性沙门菌SYBR GreenⅠ实时荧光定量PCR检测方法的标准曲线方程为y=-3.397 1 x+27.313,相关系数r2=0.999 6。该方法的灵敏度为5×100copies/μL,是普通PCR(5×102 copies/μL)的100倍。对10种常见食源性细菌的检测结果均为阴性;组内和组间重复试验的变异系数均低于1%。对90份肉品样品的检出率(6/90,6.67%)显著高于快速MALDI-TOF-MS法和传统的细菌分离鉴定方法(均为3/90,3.33%)。结果表明,本试验建立的致病性沙门菌SYBR GreenⅠ实时荧光定量PCR灵敏度高,且特异、稳定,适用于食品中致病性沙门菌的快速检测。     

鼠伤寒沙门菌SL1344 sseK2基因的克隆表达及生物信息学分析

为研究鼠伤寒沙门菌SL1344株SseK2蛋白的生物学特性,通过PCR扩增sseK2基因,构建原核表达质粒pET-32a-sseK2,经大肠杆菌BL21(DE3)表达、纯化后经SDS-PAGE和Western-blot鉴定,对sseK2基因编码的氨基酸进行生物信息学分析。结果显示,鼠伤寒沙门菌SseK2蛋白在大肠杆菌中呈可溶性表达,纯化获得较高纯度的蛋白。生物信息学分析显示,SseK2蛋白为可溶性蛋白,由348个氨基酸组成,分子质量约39.57 ku。该蛋白不含信号肽、无跨膜区,为膜外蛋白,包含4个N-糖基化位点、3个O-糖基化位点、56个磷酸化位点、15个B细胞线性结合位点和8个T细胞结合位点以及10个二硫键。其蛋白二级结构中以α-螺旋(Hh)占33.05%,无规则卷曲(Cc)占27.30%为主。成功构建了原核表达载体,并对其进行生物信息学分析,为进一步研究sseK2基因的生物学功能及在沙门菌致病机制中的作用奠定了良好基础。     

基于thyA基因的大肠杆菌染色体-质粒平衡致死系统的构建与应用

将大肠杆菌DH5α在含胸腺嘧啶核苷和三甲氧苄二氨嘧啶的琼脂平板上培养,获得了遗传稳定的ThyA-大肠杆菌DH5α21株。用PCR扩增野生菌和ThyA-大肠杆菌的thyA基因,将测定的序列与野生型thyA基因相比,6号突变株的thyA基因第92位碱基发生G→A转换,导致第31位甘氨酸被天冬氨酸替换,4、7和13号突变株的thyA基因从第73位连续缺失6个碱基,导致第25位甘氨酸和第26位苏氨酸缺失。经PCR扩增的包括启动子在内的鼠伤寒沙门菌thyA基因与野生型大肠杆菌DH5αthyA基因的同源性为86%。以鼠伤寒沙门菌thyA基因为选择标记,构建了含人溶菌酶基因的真核表达载体pDTALYZ,以ThyA-大肠杆菌为受体菌,构建了染色体-质粒平衡致死系统。在相同条件下,pDTALYZ转化ThyA-大肠杆菌的效率高于以卡那霉素抗性基因为选择标记的pcDNAKLYZ转化野生大肠杆菌的效率;在发酵培养过程中,两种重组菌的生长速率相似,质粒丢失率分别为25%和10%,湿菌重分别为57.68 g/L和51.00g/L,重组质粒产量分别为134.9 mg/L和135.0 mg/L。     

稳定携带TAT-Apoptin融合基因的重组减毒鼠伤寒沙门菌的构建及其基本生物学特性的测定

为构建能稳定携带TAT-Apoptin融合基因的重组减毒鼠伤寒沙门菌,以pMD19-T-Apoptin为模板,PCR扩增出TAT-Apoptin融合基因,将其连接至pYA3493,构建了重组质粒pYA3493-TAT-Apoptin;然后将其电转至减毒鼠伤寒沙门菌SL1344ΔcrpΔasd,并对该重组减毒沙门菌的生长特性、遗传稳定性和表达特性进行了测定。PCR和测序结果表明,成功构建了重组质粒pYA3493-TAT-Apoptin。进一步对重组减毒鼠伤寒沙门菌SL1344ΔcrpΔasd(pYA3493-TAT-Apoptin)研究发现,其生长速度比强毒株SL1344明显减慢,与缺失株SL1344ΔcrpΔasd及互补菌株SL1344ΔcrpΔasd(pYA3493)基本一致,且能够稳定遗传TAT-Apoptin融合基因。重组菌培养上清经SDS-PAGE能检测到TAT-Apoptin蛋白条带。以上结果证明,能稳定携带TAT-Apoptin融合基因的重组减毒鼠伤寒沙门菌SL1344ΔcrpΔasd(pYA3493-TAT-Apoptin)构建成功,且该重组菌能分泌性表达TAT-Apoptin蛋白。     

猪细小病毒Z株NS部分基因的扩增及序列分析

从猪细小病毒Z株提取基因组DNA ,利用PCR扩增部分基因 ,并对该扩增片段进行测序与分析。结果表明 ,扩增的NS基因长 330bp ,编码 10 9个氨基酸。氨基酸序列中含有猪细小病毒的重要保守序列 ,并有 1个潜在的糖基化位点NFSN。Z株NS基因与其他猪细小病毒Kresse、NADL2 2、NADL2 1株的核苷酸同源性分别为 99%、98%、98% ,氨基酸同源性均为 99%。     

Inter-Korean relations and the roles of the US and of the EU

The North Korean nuclear standoff has escalated since North Korea let slip that it was developing nuclear weapons while James Kelly, U.S. Assistant Secretary of East Asian and Pacific Affairs, was visiting Pyongyang in October 2002. The Bush administration has acknowledged that the United States will not discount a preemptive military strike on the suspected nuclear development facilities, and North Korea has stood firm against any pressure to halt its nuclear development from the international community. Dialogue and meetings for international cooperation among the major countries concerned, including Korea, the U.S., Japan and China, have been activated to resolve the nuclear crisis in the Korean Peninsula, especially after the U.S. pronounced an end to its war against Iraq in early May of this year. In the middle of the rapidly changing situation on the Korean peninsula, the inter-Korean reconciliation and cooperation initiated by the Sunshine Policy of the Kim Dae Jung administration and followed by the Roh Moo-hyun administrations Policy of Peace and Prosperity are not separable from the nuclear situation. This paper follows recent developments of inter-Korean relations, illustrates South Koreas stance on the issue and suggests the EUs role on the nuclear issue. This is a revised version of the paper presented at the Asia-EU Journalists Seminar organized by the Asia Europe Foundation and the Korea Press Foundation (KPF), held in Brussels, Belgium, May 22 2003. Financial support from the KPF is gratefully acknowledged.     

海陆复合型地缘政治大国的战略选择与崛起成败

国际体系是一个复杂的利益交互系统,在系统中存在着众多战略层面的施动—反馈模式。地缘政治大国的安全战略选择决定了它与体系其他主要成员间的互动方式,互动方式决定了其可能面临的结构性压力,并最终影响到大国崛起的兴衰成败。通过本项研究可以发现:海陆复合型崛起大国的战略模式可以分为"区域陆权"战略、"全球海权"战略和"区域/全球海陆并举"战略。如果崛起大国尚未形成稳固的陆基周边环境,那么追求"区域陆权"战略是最优选择;如果拥有稳固陆基周边环境,那么追求"全球海权"战略是最优选择。而"海陆并举"战略因"同色竞争"原理,既可能同周边国家陷入"区域陆权"优势的安全困境,也可能同"全球海权"国家陷入争霸战争。因此,"海陆并举"战略往往容易造就一个反对自身崛起的海陆权力联姻。此外,追求单一的"差色互补"原理容易实现海陆功能分异背景下的战略结盟,进而影响大国崛起战略的操作实施。     

The EU and Libya and the Art of the Possible

In spite of geographic proximity and a number of shared interests, the European Union and Libya have a history of strained relations. The war of 2011 provided an opportunity for a fresh start, but so far neither side has been able to reap benefits from an entirely new political situation. Instead, Libya’s difficult internal situation has not only slowed down the process of rapprochement, but also increased EU concern. At a time when cooperation becomes a necessity rather than a choice, Libya is now down-spiralling into implosion at the levels of security, bureaucracy and economy, to the point where it cannot absorb the offers being made.     

从斯大林功过看苏联兴亡——评《苏联兴亡史论》

一　对斯大林功过的评价要与时俱进要考察苏联兴亡的历史进程、总结苏联兴亡的历史经验 ,首先就要探究从列宁到戈尔巴乔夫苏联历届党政首脑的理论、路线、体制和实践的演变问题 ,尤其是涉及对斯大林功过的评价问题。因为斯大林执政时间最长 ,而且苏联的社会主义制度主要是在斯大林执政的 3 0年间( 1 92 3～ 1 95 3年 )建成并且得到巩固的 ,随后 3 0多年苏联斯大林模式没有发生什么大变化。如果我们肯定斯大林是“功大于过”,那么就要肯定苏联的社会主义模式基本上是成功的、优秀的 ,尽管难免还有一些弊端 ,那只要经过小修小补就能更加显示优…     

Campaign Advertising and the Stimulation and Activation of the Authoritarian Personality

This research examines whether authoritarianism can be stimulated and activated by politicians. The traditional belief is that psychological traits are basically quasipermanent structures that consistently determine behavior, but newer research suggests that these traits can be stimulated. This research tests whether campaigns can stimulate traits with targeted messages. I do so by exposing subjects in an experiment to political television advertising that was designed to stimulate known correlates of authoritarianism, such as fear. The results show that authoritarianism is stimulated in treatment groups that watched advertising designed to invoke threat and the strong-father metaphor and the treatment effect is greater on conservatives. I also show that watching these commercials leads to an activation of authoritarianism that influences its predictive power over support for torture. This suggests that politicians can use emotional appeals to stimulate advantageous personality traits, and that these ads also influence the public's attitudes through activation.     

美洲国家首脑会议与美洲自由贸易区的前景

自1994年至今共举行了4届美洲国家首脑会议,每届会议均与美国所倡议的美洲自由贸易区有密切关系。2005年11月在阿根廷举行的第四届首脑会议上,美洲国家围绕着是否重启美洲自由贸易区谈判问题产生分歧。一些拉美国家对在香港举行的WTO第六次部长级会议的成果不甚满意。美洲自由贸易区的前景不容乐观。     

China and the WTO: the theory and practice of compliance

Since China joined the World Trade Organization (WTO) in December2001, attention has turned to the issue of whether or not Chinais a responsible member of the organization and how compliantChina is with WTO rules. This article discusses the difficultiesfaced by China, as a responsible rising power, in trying toadjust itself to global trading norms. It examines the theoryof compliance in international relations from the perspectivesof neo-realism, liberal institutionalism and social constructivism,and then tests these perspectives by examining the mechanismsused to gauge China's compliance, both bilaterally by the UnitedStates and multilaterally through the Dispute Settlement Mechanismand the Transition Review Mechanism of the WTO. The result ismixed: different opinions exist as to how compliant China hasbeen but, on the whole, most monitors agree that China has triedhard to comply with WTO requirements in various areas, thoughmuch remains to be done. The most severe tests will come inthe next few years when China's financial and service sectorswill have to face fundamental changes to the way they operate.     

Globalization and the Reform of the International Financial and Monetary Systems

The outbreak of the global financial cdsis has called into question U.S.-style ＂financial capitalism.＂ Protectionism, currency multilateralism, decentralization of financial decision-making power and the nationalism of resources are gaining ground. The emerging economies are on the rise. The world power order is becoming multi-polar. Relations between countries are growing more diverse. The global governance model is undergoing fundamental changes. Global governance mechanisms, which are more representative and reflective of the diverse interests （such as the G20 and the UN climate change conference）, along with a reform of the international monetary system, will also help shape the future world order.     

离岸市场的发展与人民币国际化的推进

人民币国际化自2009年正式启动以来,在跨境贸易结算等领域取得了很大进展,人民币离岸市场的建设也取得了一定成绩,但目前来看并没有充分发挥预期中的功能,这也引发了各界对人民币国际化模式的进一步讨论。原则上,由于我国资本项目尚未完全开放,离岸市场有助于克服人民币自由流动受限这一障碍。历史经验也表明,在一定条件下离岸市场能够有效地推进货币国际化。人民币国际化应当遵循贸易结算、离岸市场、资本输出、跨国企业四者并举的"中国模式",其中未来的离岸市场发展需要相应的条件和配套准备才能真正为人民币国际化提供引擎。     

Uneven and combined confusion: on the geopolitical origins of capitalism and the rise of the west

This article offers a critique of Alexander Anievas and Kerem Ni?ancio?lu’s How the West came to rule: the geopolitical origins of capitalism. We argue that while all historiography features a number of silences, shortcomings or omissions, the omissions in How the West came to rule lead to a mistaken view of the emergence of capitalism. There are two main issues to be confronted. First, we argue that Anievas and Ni?ancio?lu have an inadequate and misleading understanding of “capital” and “capitalism” that tilts them towards a theoretical stance that comes very close to arguing that everything caused capitalism while at the same time having no clear and convincing definition of “capital” or “capitalism”. Second, there are at least three omissions—particular to England/Britain within a geopolitical context—that should be discussed in any attempt to explain the development of capitalism: the financial revolution and the Bank of England; the transition to coal energy; and the capitalization of state power as it relates to war, colonialism and slavery. We conclude by calling for a connected-histories approach within the framework of capital as power.