鸡毒霉形体TM-1基因原核表达载体的构建及表达

对从含有鸡毒霉形体H3株TM 1基因的大肠埃希氏菌DH5α中提取的质粒,进行EcoRⅠ和SalⅠ双酶切,获得了大约为786bp的目的片段。将此片段与经EcoRⅠ和SalⅠ双酶切的线性原核表达载体pET 30a 1连接,转化宿主菌BL21(DE3),筛选阳性克隆。提取质粒,经酶切、PCR扩增和测序分析确证其插入正确,从而构建成了重组表达载体。阳性克隆用IPTG诱导表达,表达产物经SDS PAGE电泳分析,证明其分子质量约为29ku;Western blotting分析表明,该蛋白可被鸡毒霉形体阳性血清识别,具有生物学活性。     

新城疫病毒HN基因与T4噬菌体SOC基因在大肠埃希氏菌中的融合表达

用RT PCR方法扩增了新城疫病毒 (NDV)的完整HN基因序列 ;设计了 1对带有EcoRⅠ限制位点的引物 ,用PCR扩增出了HN基因片段。将该片段克隆至 pSOC质粒SOC基因 3′端 ,成功构建了重组质粒pSOC HN。用该重组质粒转化E .coliBL 2 1(DE3)感受态细胞 ,以终浓度 1mmol/mL的IPTG诱导表达。在SDS PAGE凝胶上可检测到分子质量约 6 7ku的特异条带 ,蛋白质印迹法证实 ,表达产物与NDV抗血清具有良好的反应性     

猪胸膜肺炎放线杆菌ApxⅣA 2.5kb基因片段的分子克隆及其表达

以猪传染性胸膜肺炎放线杆菌血清1型国内分离株721株基因组DNA为模板,用PCR方法扩增出ApxⅣA基因2.5kb特异片段,将其克隆于pMD18T中,经酶切鉴定筛选重组质粒后进行核苷酸序列测定,并与GenBank中登录的血清1型ApxⅣA(AX002405)基因进行比较,结果显示核苷酸同源性为99.5%。将该片段亚克隆到原核表达载体pGEX6P1的EcoRⅠ/SalⅠ位点,成功地构建了重组表达载体pGEXapxⅣA,并转化大肠埃希氏菌BL21,获得了表达。SDSPAGE检测结果显示,表达的融合蛋白分子质量约为117ku,与预期片段大小相符;Westernblotting分析证实,该融合蛋白具有免疫学活性。     

亚洲Ⅰ型口蹄疫病毒VP1基因真核表达载体的构建及其免疫活性的初步分析

提取亚洲Ⅰ型口蹄疫病毒总RNA,用特异性引物通过RTPCR反应获得了约750bp的核酸片段,将其与pGEMTEasy载体连接,并转化大肠埃希氏菌JM109。重组质粒经PCR、酶切鉴定及测序,证明所克隆的片段含有完整的VP1基因;将重组质粒与表达载体pcDNA3.1( )分别用BamHⅠ和EcoRⅠ双酶切后连接,构建成重组表达质粒,转化宿主菌DH5α,筛选阳性克隆。测序结果证明,目的基因正确插入到表达载体中,命名为pcDNAV;用同样方法将IL18基因插入pcDNAV中,命名为pcDNAVI。用pcDNAVI免疫豚鼠,2周后加强免疫1次,每周用ELISA检测豚鼠血清中的抗体水平。结果表明,pcDNAVI可引发机体产生体液免疫。     

O型口蹄疫病毒OA58株3C基因的克隆与真核表达

利用RT-PCR技术扩增获得了长度约640 bp的口蹄疫病毒目的基因,回收片段后与pMD18-T载体进行连接,测序结果表明获得了口蹄疫病毒3C基因。将重组质粒pMD18-3C与表达载体pEGFP-N1分别用BamHⅠ+XhoⅠ双酶切后,进行定向亚克隆,对重组表达质粒pEGFP-3C进行PCR、酶切鉴定及DNA测序,结果表明重组表达质粒所含的3C基因读码框正确。用pEGFP-3C转染BHK-21细胞,荧光显示目的基因EGFP发出绿色荧光;对3C转录的mRNA进行RT-PCR鉴定,证实3C基因在BHK-21细胞中得到了表达。     

堆形艾美球虫广东株3-1E基因在毕赤酵母中的表达

以重组质粒pGEM-3-1E为模板,扩增了序列两端分别含有EcoRⅠ和XbaⅠ酶切位点的堆形艾美球虫(Eimeria acervulina)广东株3-1E基因(长度为529 bp),将3-1E基因克隆至巴斯德毕赤酵母分泌型表达载体pPICZαC中,构建了酵母表达质粒pPICZαC-3-1E。转化毕赤酵母X-33得到含有3-1E基因的重组酵母,甲醇诱导产生的目的蛋白经SDS-PAGE分析和免疫印迹检测,表明毕赤酵母成功表达了3-1E基因。     

环形泰勒虫裂殖体表面蛋白基因高免疫原性区片段的克隆及其表达

根据环形泰勒虫TaA12 72 1株裂殖体表面抗原 (TaSP)基因的序列设计了 1对引物 ,用PCR扩增出该基因长为 393bp的高免疫原性区片段 (SBxp)。将扩增产物连接到 pGEM TEasy载体上 ,转化入大肠埃希氏菌JM 10 9中进行克隆。随后将重组质粒 pGEM SBxp和表达载体 pGEX 4T 1分别以BamHⅠ、XhoⅠ酶切后 ,将酶切的目的片段SBxp亚克隆到原核表达载体中构建了重组表达载体 pGEX 4T 1 SBxp ,并将其转化到BL2 1宿主菌中。提取重组质粒经BamHⅠ、XhoⅠ酶切、PCR鉴定和测序确证后 ,阳性克隆用IPTG诱导表达。收集不同时间的菌液进行SDS PAGE电泳和Westernblotting检测。结果 ,SBxp基因在大肠埃希氏菌中成功表达 ,其表达产物为分子质量 4 3ku的融合蛋白 ,该产物能被牛环形泰勒虫阳性血清所识别。     

猪日本乙型脑炎病毒NS1基因重组腺病毒的构建及表达

应用RT-PCR方法扩增出猪日本乙型脑炎病毒(JEV)的NS1基因,通过SalⅠ EcoRⅠ双酶切后把该基因插入到经过同样双酶切的穿梭质粒pDC315中。重组穿梭载体经过双酶切和PCR鉴定后进行测序,序列测定正确。将获得的重组穿梭质粒与腺病毒骨架质粒共转染293细胞后获得重组腺病毒。PCR鉴定及间接ELISA检测的结果证明所构建的重组腺病毒成功地表达了JEV的NS1。     

产气荚膜梭菌贵州分离株α-毒素基因的克隆与鉴定

提取了A型产气荚膜梭菌贵州分离株的染色体DNA,经聚合酶链反应(PCR)扩增并回收获得了1条242bp的特异性片段,将该基因片段克隆到质粒载体pUC19中,并转化至大肠埃希氏菌JM109中;经氨苄青霉素(Amp)抗药性和显色反应筛选及重组质粒酶切分析、PCR鉴定和Southern blotting检测,表明该重组质粒是产气荚膜梭菌α 毒素基因的克隆。     

亚洲Ⅰ型口蹄疫病毒VP1基因重组犬2型腺病毒的构建

应用PCR方法扩增出亚洲Ⅰ型口蹄疫病毒的VP1基因,继而构建出真核表达载体pVAX-VP1。再将含有巨细胞启动子CMV的目的基因表达盒连接到穿梭载体pVAX△E3上,筛选出VP1表达盒方向与E3区转录方向一致的重组子,获得转移载体。再经酶切,连接等方法获得重组腺病毒质粒。利用脂质体介导将重组质粒转染DK细胞,转染3次后,细胞出现明显的腺病毒病变。经PCR扩增、测序检测及基因组酶切鉴定,证明该病毒即为重组犬2型腺病毒。命名为CAV2/FMDV。经验证,该重组毒可稳定遗传,其毒价为103.5TCID50/mL。     

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.     

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.     

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.     

My Journey to Artistic Peak

正I was born in a doctor’s family in a village in Heze,Shandong Province.Since the ancient times,Heze has been home to many painters and literature masters due to the influence of Confucianism.My grandfather and greatgrandfather were both good at calligraphy,not least to mention their medical skills.Even by now,senior people in our village still praise them for it.Both of them had deceased before I was born.     

Sidelights on Australian and New Zealand's Rotary Club Delegation's Visit to China

正At the invitation of the Chinese Association for International Understanding(CAFIU),a 6-member Australian and New Zealand’s Rotary Club delegation visited China from September 23 to September 27.As a staff member of CAFIU,I had the honor to take part in the reception of the delegation throughout this trip.