猪流行性腹泻病毒重组S1蛋白的免疫原性评价

将猪流行性腹泻病毒(PEDV)流行毒株S1基因经密码子优化后克隆到慢病毒表达载体,在293T细胞包装PEDV S1重组慢病毒;重组慢病毒感染293T细胞,经有限稀释法筛选获得高效稳定表达S1蛋白的重组细胞系HEK-293T-S1;生产、纯化S1蛋白,添加佐剂制备成重组S1亚单位疫苗,经肌肉注射免疫实验用巴马小型猪,评价...     

慢病毒载体介导稳定表达pAPN的Vero细胞系的构建及应用

将完整的pAPN序列通过同源重组克隆入慢病毒表达载体pLVX-mCherry,并将该重组质粒pLVX-mCherry-pAPN与慢病毒包装质粒pLP1、pLP2、pLP-VSVG共转染293T细胞,在293T细胞中包装成含目的基因的重组慢病毒。将收获的重组病毒感染Vero细胞,经过嘌呤霉素筛选和细胞有限稀释法,筛选出表达pAPN的细胞。通过RT-PCR和间接免疫荧光试验(IFA)证明了所获细胞株能够稳定表达pAPN蛋白,命名为Vero-APN-B6。IFA试验和TCID50试验结果表明:与Vero细胞相比,Vero-APN-B6更能促进猪流行性腹泻病毒(PEDV)的增殖。利用该细胞株成功地从猪小肠组织中分离到了PEDV SC-MS株;经过鉴定该毒株为变异毒株。     

慢病毒介导的bta-miR-222稳定过表达MDBK细胞株的构建及鉴定

拟获取稳定表达bta-miR-222的MDBK细胞株,为后续探索bta-miR-222在MDBK细胞中的作用奠定基础。本研究使用PCR技术,从MDBK细胞基因组与pEGFP-N1质粒中扩增pri-bta-miR-222和GFP片段,胶回收,先克隆于pMD18-T载体中,转化入大肠杆菌,酶切鉴定及测序正确,再克隆于pCDH-CMVMCS-EF1-Puro载体,构建含bta-miR-222基因的miRNA慢病毒重组质粒pCDH-miR-222-GFP。将pCDHmiR-222-GFP与两个包装质粒PSP、PMD共转染293T细胞,制备含bta-miR-222基因的重组慢病毒V_(PCDH+miR-222)。用制备成功的慢病毒V_(PCDH+miR-222)感染MDBK细胞,经Puromycin嘌呤霉素筛选,荧光显微镜观察感染效率。结果显示,pCDH-miR-222-GFP过表达质粒构建成功,序列比对符合率达100%。将pCDH-miR-222-GFP、PSP和PMD共转染后,通过荧光显微镜观察到293T细胞分布大量的荧光,说明成功构建了重组慢病毒V_(PCDH+miR-222),经Puromycin嘌呤霉素筛选10代后,V_(PCDH+miR-222)在MDBK细胞中仍具有感染效率。本研究成功获取稳定表达bta-miR-222的MDBK细胞株,为进一步研究bta-miR-222在病毒感染MDBK细胞中的功能奠定了基础。     

稳定表达日本脑炎病毒NS2B蛋白的细胞系的建立

本研究旨在建立能稳定表达日本脑炎病毒NS2B蛋白的细胞系。利用RT-PCR扩增NS2B基因,并将之克隆至带嘌呤霉素抗性基因的慢病毒载体Psin-EF2-MCS-Pur中。然后用慢病毒载体系统包装慢病毒,并将之转导正常293T细胞,通过嘌呤霉素加压筛选纯化细胞系。最后利用Western-blot、间接免疫荧光试验等方法对NS2B蛋白在细胞系中的表达情况进行了鉴定。结果表明,NS2B蛋白可以在所建立的细胞系中被稳定、高效地表达。在此基础上,利用该细胞系,通过串联亲和层析与质谱技术,筛选出了与NS2B可能相互作用的宿主蛋白,从而为深入阐明NS2B的功能及其与宿主细胞相互作用机制的研究奠定了基础。     

H3N2亚型猪流感病毒反向遗传操作技术平台的建立

为了构建猪流感病毒A/swine/Heilongjiang/1/2005(H3N2)的反向遗传操作技术平台,利用RT-PCR扩增了H3N2亚型猪流感病毒的8个基因片段,分别连接到pBD双向转录表达载体上。将8个重组质粒纯化后,共转染293T细胞,48h后收集上清,接种MDCK细胞。当MDCK细胞呈现明显病变时,收集上清,做血凝试验,检测出有血凝现象。通过MDCK细胞病变情况、生长曲线、空斑形态等验证拯救病毒的可靠性,结果发现:野生毒株和拯救毒株在MDCK细胞上引起的病变情况基本一致,生长曲线的测定结果没有明显差异,空斑形态也极其相似。H3N2亚型猪流感病毒的成功拯救为猪流感病毒的致病机理、传播机制、基因功能研究以及疫苗研发奠定了基础。     

猪瘟病毒T细胞表位与猪细小病毒VP2融合基因在重组杆状病毒中的表达

将猪细小病毒分离株LJL12 VP2基因和猪瘟病毒多肽基因E290克隆至真核表达载体pMel Ba-cA,将该重组质粒与Bac-N-Blue DNA共转染昆虫细胞,并对表达产物进行分析,构建了表达猪瘟病毒T细胞表位和猪细小病毒VP2融合蛋白的重组杆状病毒。结果显示,获得了含VP2基因和E290基因的重组质粒pM-VP2-E290,昆虫细胞sf9的表达产物经SDS-PAGE、Western-blot检测后确定所表达的蛋白质分子质量大小约为67 ku,且具有天然蛋白的抗原特性。免疫电镜观察可见表达产物形成的病毒样颗粒。证实,成功构建了同时含有PPV VP2基因和CSFV特异性T细胞表位基因的重组杆状病毒,并在昆虫细胞中得到了高效表达。     

传染性喉气管炎病毒王岗株gB基因gC基因和gD基因的真核表达

将传染性喉气管炎病毒(ILTV)王岗(WG)株的gB、gC、gD基因分别克隆到真核表达载体pCAGGS的EcoRⅠ位点,经过酶切、测序分析,筛选鉴定出含有gB、gC、gD基因的重组质粒,分别命名为pCAGGgB、pCAGGgC、pCAGGgD。用质粒纯化试剂盒对重组质粒进行了纯化,将纯化后的质粒转染293T细胞,用间接免疫荧光技术检测了目的蛋白的表达情况。检测结果表明,目的蛋白得到了真实的表达。     

稳定表达人TMPRSS2基因BHK细胞系的建立及其对新城疫弱毒增殖效率评价

为提高细胞培养系统中新城疫病毒的增殖效率,通过构建TMPRSS2过表达的BHK细胞系,探索其对新城疫弱毒增殖的影响.本研究将TMPRSS2基因克隆于噬菌体载体pHAGE中,构建重组质粒pHAGE-TMPRSS2.将重组质粒pHAGE-TMPRSS2以及辅助包装质粒psPAX2和pMD2G共转染至293T细胞并收集上清....     

猪Hsp40基因过表达与干扰重组慢病毒载体的构建

热休克蛋白40(Hsp40)作为Hsp70蛋白的分子伴侣,在病毒生命周期中扮演重要角色,笔者拟通过慢病毒感染技术分别建立Hsp40基因过表达和沉默的猪肺泡巨噬细胞系,为以后开展猪Hsp40蛋白调控猪圆环病毒2型(PCV2)复制的机制研究提供有效模型。提取猪肺泡巨噬细胞(PAM)的总RNA,反转录成cDNA后用于Hsp40基因编码区的扩增,并将其克隆到慢病毒过表达载体pCDH-CMV-MCS-EF1-GreenPuro。测序正确后,设计针对Hsp40基因的4条shRNA干扰序列和1条阴性对照序列,并将其分别插入到慢病毒干扰载体pCDH-U6-MCS-EF1-GreenPuro。将上述6个载体分别与3个辅助载体pVSV-G、pRev和p Gag/Pol共转染293T细胞进行病毒包装,测定其滴度后用于感染PAM细胞,采用RT-qPCR和Westernblot方法对Hsp40的表达效果进行鉴定。猪Hsp40基因过表达和沉默重组慢病毒的获得为进一步研究Hsp40蛋白对PCV2复制的影响和机制奠定了基础。     

西尼罗病毒E蛋白的原核表达及其多克隆抗体的制备

利用PCR技术扩增西尼罗病毒(WNV)E蛋白基因并将其插入原核表达载体pET-30a(+),构建重组质粒pET-30a(+)-WNV-E,并转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导目的蛋白表达,对诱导条件进行优化,利用His镍柱亲和层析法纯化目的蛋白,纯化后的蛋白经SDS-PAGE与Westernblot鉴定正确后,免疫BALB/c小鼠制备多克隆抗体并进行间接免疫荧光鉴定。结果显示:WNV E蛋白在大肠杆菌中以包涵体形式被成功表达,蛋白最佳诱导条件为37℃,0.8 mmol/L IPTG诱导5 h,经间接免疫荧光鉴定多克隆抗体能特异性识别WNV E蛋白。本研究成功表达并纯化了WNV E蛋白,证明该蛋白具有良好的免疫原性,为WNV抗原、抗体检测方法的建立和新型疫苗的研发及相关研究奠定了基础。     

China＇s Rise vs. International Order Evolution

A heated debate over the rise of China has been going on unabated in the international community for nearly two decades. However, with the elapse of time, the initial divergence of views in the mainstream opinion has gradually diminished and the gap in China policy among nations has likewise narrowed down. Instead, reason and a realistic approach have eventually taken center stare.     

The U. S. Policy toward DPRK: Opportunities and Constraints

布什政府上台以来,始终未能阻止朝鲜拥核步伐.朝核试后,美对朝政策有了新的调整,六方会谈得以复会.但美朝双方受各自国家利益和政策目标的限制,在朝鲜半岛无核化问题上的尖锐分歧难以解决,朝核危机仍存在再度升级的可能.     

Marxist Philosophy vs. International Relations Research in China

This article argues for the confirmation of Marxist philosophy as the ideology that guides international relations （IR） studies in China. Justification comes through the following points. According to Marxism, correct theoretical guidance serves as the prerequisite for correct practice. Marxist philosophy embodies, by nature, righteousness, in opposition to political hegemony. Accordingly, practice under its guidance represents fruitful revolutionary realism. Finally, advanced studies of international relations in this country cannot be separated from philosophical thinking. Using these arguments as background, this article specifically advocates materialism, patriotism, and service to the people as the corner-stone of China＇s IR theory building.     

The Development of EU-Russian Relations

The EU＇s strategy for Russia since the end of the cold war has to a large extent failed, and the direct consequences are the currently deadlocked EU-Russia relationship. There are three reasons for this： the EU and Russia＇s respective strength and status relative to each other have been changing since the beginning of this century; the EU＇s strategy towards Russia has been more fragmenting in recently years; and the American factor. The EU-Russia relations could continue to be in poor shape in the years to come, and it is very likely sometimes even further worsening. But the two sides would refrain from extreme measures and prevent their relations from sliding into real cold war.     

Excavating Concealed Tradeoffs: How Russians Watch the News

Citizens in democracies are expected to make better decisions if they understand policy tradeoffs. However, politicians rarely have incentives to communicate them; citizens are uncomfortable choosing among valued outcomes; and devising a common metric is difficult. It is not surprising that in the United States the environment provides relatively little cuing or priming of tradeoffs in television news. Russian citizens, on the other hand, face a media environment in which tradeoff cuing is intentionally suppressed by owners' agendas, yet viewers detect concealed tradeoffs even in the absence of tradeoff priming and viewpoint diversity. Analysis of discourse among ordinary Russians in 16 focus groups convened in four cities, differentiated by political reform and media market environments, showed that when watching news in which tradeoffs are thoroughly concealed, viewers challenge stories by offering a broad spectrum of uncued tradeoffs. Tradeoffs come from diverse policy domains and represent a range of cognitive strategies, some of which are considerably more abstract than others and link elements of their observations and assumptions (together with what they can extract from the stories) into complex reasoning outcomes.     

The U.S. Global Strategy as Seen from the Debate in the Primary Election

There may be more than one year to go before the 2008 U.S. presidential election, but the primary election of the two parties has already begun ahead of time. Both the Democratic and Republican candidates for the     

Young American China Watchers＇ Views on China

In this paper, the author has made a thorough study over the new generation of American China Watchers. Most of the pages are devoted to a detailed comparison between the old generation and the young generation of American China Watchers regarding to their background, education, and views over China. At the end of the paper, the author also gives some policy suggestions for both the Chinese and U.S. government based on the findings in this study.     

The Balance Between Coercion and Accommodation

1. Introduction
On the morning of April 1st, 2001, a U.S. EP-3 surveillance aircraft was conducting an intelligence flight along the Chinese territorial border in the airspace south-east of China＇s Hainan Island. Two Chinese fighter jets intercepted the spy plane and followed its flight, as is considered ordinary international practice. For reasons unknown, the EP-3 airplane made a sudden maneuver and crashed into one of the Chinese planes. The Chinese plane dropped into the sea, the pilot missing. The U.S. spy plane was also severely damaged. It intruded into Chinese territorial space and landed on Hainan Island without official permission from the Chinese side. After landing, the spy plane and 24 crew on board were detained by the Chinese authorities. This air collision episode was the first of its kind in the China-U.S. relations and an urgent diplomatic crisis ensued.     

Constitution of the Chinese Associa tion for Interna tiona l Understa nding(Approved by the Plenary Session of the Eleventh Executive Council on April 2, 2014)

正Chapter I General Rules Article 1 The Chinese Association for International Understanding is a national non-governmental organization(NGO)in the People’s Republic of China.Article 2 The official name of the Association is"Zhong Guo Guo Ji Jiao Liu Xie Hui"in Chinese,or"Jiao Liu Xie Hui"for short.Its English translation is"Chinese Association for International Understanding",and abbreviated as"CAFIU".     

Guide to Authors

正Published by China Institute of International Studies,China International Studies is China’s first English-language journal on diplomacy and international politics for formal circulation.The magazine mainly publishes the latest research achievements of Chinese scholars and specialists on China’s diplomacy,international politics and international relations under such columns as China’s Diplomacy,International Relations,International Politics,International Security,Regional Issues and World Economy.