藏猪IGF-1成熟肽真核表达载体的构建及其在毕赤酵母GS115中的表达

构建藏猪IGF-1成熟肽基因的真核表达载体,通过毕赤酵母表达系统获得具有生物活性的IGF-1成熟蛋白。根据GenBank中猪IGF-1基因设计1对引物,以构建的藏猪pMD19-T-IGF-1重组质粒为模板,克隆IGF-1成熟肽基因,并将其克隆于表达载体pPIC9k,获得重组穿梭质粒pPIC9k-IGF-1,电转毕赤酵母GS115感受态,对重组酵母转化子经MM、MD平板筛选和PCR鉴定后,经G418抗性梯度筛选,获得多拷贝重组菌株,甲醇诱导表达后进行Tricne-SDS-PAGE和Dot blot分析。Tricne-SDS-PAGE和Dot blot分析显示,表达的目的蛋白分子质量约为13 ku,获得成功表达;CCK8法检测显示,纯化后的IGF-1重组蛋白能够促进细胞增殖。表明,本研究成功构建了藏猪IGF-1成熟肽真核表达质粒pPIC9k-IGF-1,并在毕赤酵母GS115中得到了有效表达,表达产物具有促进细胞增殖的生物活性。     

猪带绦虫TSO18基因真核表达载体的构建及其表达

采用PCR从重组克隆载体pGEMTSO18中扩增出猪带绦虫六钩蚴TSO18基因,与毕赤酵母分泌性表达载体pPIC9k相连接,构建重组表达载体pPIC9kTSO18,转化大肠埃希氏菌JM109,经测序证实,基因序列完全正确。制备重组质粒pPIC9kTSO18,用SalⅠ线性化,并电转化毕赤酵母(Pichiapastoris)GS115,使重组表达载体与酵母染色体发生同源整合。采用G418抗性梯度法筛选得到高拷贝重组菌株,以甲醇进行诱导表达,SDSPAGE和Westernblotting分析结果表明,诱导表达的培养上清中表达出具有反应活性的16ku重组蛋白,目的蛋白约占培养上清液中蛋白总量的80%以上,诱导72h目的蛋白表达量为0.5mg/mL。     

家蝇幼虫溶菌酶1基因的克隆与真核表达及表达产物抑菌活性的检测

采用PCR扩增获得家蝇溶菌酶1基因(Musca domesticalysozyme-1,MdL-Ⅰ),构建真核重组表达质粒pPIC9K-MdL-Ⅰ。将线性化的重组质粒电击转入巴斯德毕赤酵母感受态GS115细胞内。将PCR鉴定为阳性的重组酵母菌进行甲醇诱导表达,并采用Tricine-Tris-SDS-PGAE检测表达产物的大小;经镍柱亲和层析纯化获得纯度较高的重组蛋白,采用管碟法检测重组蛋白的生物学活性。结果显示,重组质粒pPIC9K-MdL-Ⅰ获得高效表达,蛋白质的分子质量为14.2ku,管碟法检测重组蛋白对大肠杆菌、雏沙门菌均具有体外抑菌活性。本研究为利用毕赤酵母表达系统规模化生产具有抑菌活性的MdL-Ⅰ蛋白奠定了基础。     

土耳其斯坦东毕吸虫磷酸丙糖异构酶基因的克隆及在毕赤酵母中的表达

利用RT-PCR技术从土耳其斯坦东毕吸虫(Orientobilharzia turkestanicum)成虫总RNA中扩增磷酸丙糖异构酶基因(TPI),鉴定后将目的片段与毕赤酵母表达载体pPIC9k连接,构建重组表达质粒pPIC9k-TPI,并将其电击转化到毕赤酵母GS115中,重组菌株经甲醇诱导后表达的TPI蛋白,经SDS-PAGE、western-blotting检测,并利用葡聚糖凝胶层析柱纯化。结果显示,成功地克隆了土耳其斯坦东毕吸虫TPI；重组毕赤酵母表达了分子质量为43 ku的TPI蛋白；葡聚糖凝胶层析过滤得到单一的TPI蛋白。     

猪囊尾蚴半胱氨酸蛋白酶TsCL-1基因在毕赤酵母中的表达

采用RT-PCR方法从猪囊尾蚴总RNA中扩增出TsCL-1基因并构建了真核表达载体pPIC9K-TsCL-1,pPIC9K-TsCL-1电转化毕赤酵母GS115.采用G418抗性梯度筛选多拷贝重组菌株,经甲醇诱导表达,应用SDS-PAGE和Western-blot分析重组蛋白的表达情况.结果显示,TsCL-1与GenBank登录的TsCL的核苷酸和氨基酸的同源性分别为99.8%和100%,与肝片吸虫和大片吸虫组织蛋白酶氨基酸的同源性为76.3%.TsCL-1基因在毕赤酵母中成功表达,表达产物的分子质量为42 ku左右;Western-blot分析表明,表达产物具有反应原性.     

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。     

微小隐孢子虫类钙调蛋白基因真核表达质粒的构建及在Hela细胞中的表达

为了构建微小隐孢子虫类钙调蛋白(calmodulin-like protein,CML)基因的真核表达质粒,并在Hela细胞中实现表达,以微小隐孢子虫卵囊cDNA为模板,通过PCR扩增CML基因,插入到克隆载体pMD18-T中。对经鉴定的pMD-CML重组质粒进行双酶切,将目的基因连接到经同样内切酶双酶切的真核表达载体pVAX1上。重组质粒经双酶切分析和测序鉴定后,用FuGENE HD转染试剂介导的方法,将重组表达质粒转染Hela细胞,用Western-blot技术和间接免疫荧光法检测外源基因的表达。结果显示,成功构建了微小隐孢子虫CML基因的真核表达质粒pVAX-CML,重组质粒在Hela细胞中实现了表达,表达产物具有良好的反应原性,为研究CML的特性和功能、寻找新的防控技术奠定了基础。     

猪CD8分子α链胞外区基因片段在毕赤酵母中的表达

为了给猪CD8α分子单克隆抗体的研制提供高生物学活性的抗原,采用PCR方法从pGEM-T-pCD8α重组质粒中克隆了pCD8α分子的胞外区基因片段,构建了真核融合表达载体pPIC9K-pCD8α-ED,在毕赤酵母中进行表达,并对重组基因工程菌的发酵时间、pH值进行了优化。结果表明,表达出约20ku的目的蛋白,诱导表达的最适pH值为5.0,诱导表达的最佳时间是120h。Western-blot分析结果显示,表达产物能被鼠抗人CD8α多克隆抗体识别,在20ku左右出现目的条带,说明表达的目的蛋白具有免疫活性结构。     

A群猪轮状病毒G5型OSU株VP7基因的克隆及其重组杆状病毒的鉴定

利用反转录-聚合酶链反应(RT-PCR)扩增出A群猪轮状病毒(PRV)G5型OSU株长度约为1.0kb的基因片段,将其克隆到pMD 18-T载体上进行测序鉴定,证明为该PRV的VP7蛋白基因,与已发表的该PRV VP7基因序列的同源性为99.8%.将该基因黏端克隆至表达载体PblueBAChisC质粒中,酶切及PCR鉴定表明获得了PRV-VP7蛋白基因与PblueBAChisC质粒的阳性重组子.纯化该重组质粒并与线性杆状病毒DNA分子Bac-N-Blue共转染昆虫细胞sf9,5 d后收获重组病毒.通过对重组杆状病毒DNA分子的酶切及PCR鉴定,确定PRV-VP7基因已正确插入.将经3次蚀斑筛选纯化后的该重组杆状病毒接种于sf9细胞大量增殖后,获得了重组杆状病毒PRV-VP7蛋白的真核表达.     

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.