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。     

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,评价重组S1蛋白的免疫原性。结果表明,疫苗经肌肉注射免疫能诱导唾液、粪便及血清产生S1特异性Ig A;证实巴马小型猪3日龄仔猪对PEDV流行毒株易感,在临床症状及组织病理学变化均符合高毒力PEDV特征,可以作为感染动物模型;在实验用巴马小型猪证实PEDV重组S1蛋白结合佐剂经肌肉注射可诱导粘膜免疫,这为PEDV基因工程疫苗研发奠定了基础。     

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。     

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。     

密码子优化对鸭坦布苏病毒E基因DNA疫苗免疫应答的增强作用

为提高DNA疫苗用于预防鸭坦布苏病毒病的免疫原性,将鸭坦布苏病毒的E基因密码子改造为鸭偏嗜的密码子,并将其作为免疫原基因克隆到pCAGGS载体中,构建真核表达质粒pCAGGS-OptiE。将pCAGGS-OptiE和先前构建的编码野生型E基因的表达质粒pCAGGS-E分别转染293T细胞,用间接免疫荧光和Western-blot检测E蛋白的表达情况。结果显示,密码子优化的重组质粒的蛋白表达量明显高于野生型质粒。将重组质粒pCAGGS-OptiE和pCAGGS-E用脂质体包裹后分别尾静脉注射免疫BALB/c小鼠,采用间接ELISA检测抗体的产生情况。结果显示,质粒pCAGGS-OptiE诱导的抗体水平明显高于pCAGGS-E,表明密码子优化的E基因能够提高蛋白的表达水平,进而增强机体免疫应答反应。     

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.     

中国需要构建怎样的周边(英文)

正The new central leadership has highlighted the strategic importance of better relations with neighboring countries and the construction of a solid foundation for China’s neighborhood strategy so as to create favorable conditions for Chinese development.1China is strengthening top-level strategy in foreign relations,focusing on global-level relations and staying committed to relations     

Learning from Others and Starting Built-to-last Companies

正Ruicheng Group was founded in Qiqihar,a city located in one of the three major ‘Chernozem belts’ in the world,the fertile Songnen Plain,and one known as the ‘home of red-crowned cranes’ Over the years,our business developed from being only one supermarket in the beginning to a corporate group operating in multiple sectors,from supermarket chain and real estate to property management and preschool education.     

中巴关系的发展及其认识误区（英文）

China and Brazil established a strategic partnership in 1993. The bilateral ties have seen rapid development in almost all areas. However, there are eight myths surrounding the China-Brazil relations. These myths are the result of a lack of mutual understanding as well as the misconceptions result misconceptions of the western observers and media. These misunderstandings have of have jeopardized images of the China-Brazil relations and also hindered further development of their bilateral relationship.     

中国的“主场外交”:机遇、挑战与任务（英文）

The successful hosting of the CICA summit has drawn great attention The attention to China's "host diplomacy". Analyzing new features of China's "host to "host diplomacy" during this new period, this paper will look at the opportunities and challenges facing China's "host diplomacy" and discuss the problems that China should address in order to do a better job in this regard and bring its "host diplomatic advantages" into full play.     

亚洲整体性崛起及其效应(英文)

Since the beginning of the 21st century, the overall rise of the Asian geographic plate has been particularly prominently. The overall rise of Asia reflects the improvement of a large number of developing countries in Asia. The long-occupied aggressive status of the United States and Europe is weakening, and the overall rise of Asia appears more highlighted, and its impact upon the world seems more intense.     

CAFIU Convenes the 11~(th) Council Meeting in Beijing

正On the morning of April 2,2014,CAFIU held the 11th Executive Council Meeting in Beijing.Present at the Meeting were Member of the Politburo of the 17th CPC Central Committee and Vice-Chairman of the 11th CPPCC National Committee Mr.Wang Gang,ViceChairman of the 11th NPC Standing Committee and President of CAFIU Mr.Zhou Tienong,ViceChairpersons of the NPC Standing Committee Mme     

Speech by Honorary President Zhou Tienong at the 11~(th) Executive Council Meeting of CAFIU (Excerpt)

正I am very pleased to attend the 11th Executive Council Meeting of CAFIU,and would like to express my hearty congratulations to President Yan Junqi,the new leadership and the newly-elected Executive Council.I also want to express my sincere thanks for choosing me to be the Honorary President of CAFIU!     

Name-list of Members of the Standing Committee of the (11)~th Executive Council (in alphabetical order of the surname's first letter)

正I.Political Parties and Government Sector Lu Yan Director,Beijing Municipal Commission of Commerce Tong Guili Member,Standing Committee of the CPC Municipal Committee of Hangzhou,Zhejiang Province Secretary,CPC Working Committee,Administrative Commission for the Sci-Tech and Innovation Industrial Park in west Hangzhou City     

FES Head of Department for International Development Cooperation Visits China

正At the invitation of CAFIU,Mme.Christiane Kesper,Director of the Department for International Development Cooperation of Friedrich-Ebert-Stiftung of Germany(FES)visited China from Oct.9 to 14.During the stay in Beijing,Mr.Li Jinjun,Vice-Minister of IDCPC and Executive Vice-President of CAFIU met with