H5N1亚型禽流感病毒NS1基因的原核表达及其ELISA检测方法的建立

采用RT-PCR技术扩增了禽流感病毒(AIV)A/Goose/HLJ/p46/2003(H5N1)的NS1基因,将其克隆于融合蛋白表达载体pMAL-c2X上,转化DH5α大肠埃希氏菌感受态细胞,经BamHⅠ和HindⅢ双酶切鉴定及序列分析,表明筛选到了重组质粒pc2X-NS1。SDS-PAGE电泳结果显示,重组质粒转化TB1大肠埃希氏菌后,经0.3mmol/L的IPTG诱导,融合蛋白MBP-NS1得到大量表达,融合蛋白以可溶形式存在,分子质量约为67ku。Western-blotting检测结果表明,融合蛋白MBP-NS1能够与H5N1亚型AIV活病毒感染康复鸭血清发生特异性反应,而不能够与H5N1亚型AIV灭活疫苗免疫鸭血清发生反应。试验初步建立了以纯化的融合蛋白MBP-NS1为包被抗原的间接ELISA检测方法,为AIV灭活疫苗免疫家禽与AIV感染家禽的鉴别诊断奠定了基础。     

H7N9亚型禽流感病毒血凝素和神经氨酸酶单因子血清的制备

为获得特异性H7N9亚型禽流感病毒(AIV)血凝素(HA)和神经氨酸酶(NA)单因子血清,根据GenBank中A/Chicken/Zhejiang/SD007/2013毒株的序列人工合成了HA基因和NA基因,然后将其分别克隆至真核表达载体pCAGGS,构建了重组真核表达质粒pCAGGS-HA和pCAGGS-NA,经酶切和测序鉴定后将阳性重组质粒转染COS-7细胞,利用H7N9AIV鸡血清进行Western-blot分析。结果显示,重组质粒中的目的基因均能够成功表达。将重组质粒以每只200μg的剂量免疫4周龄SPF鸡,二免后第14天采集血清并用间接免疫荧光试验和Western-blot检测抗体。结果显示,免疫鸡产生的血清均能与对应质粒经COS-7细胞表达的蛋白发生特异性反应。结果表明,成功制备的H7N9AIV HA蛋白和NA蛋白单因子血清具有良好的反应原性,为提高H7N9AIV的血清学检测方法的准确性提供了一定技术支撑。     

H3N2亚型猪流感病毒核蛋白基因的优化表达及抗原性分析

根据已获得的猪流感病毒(SIV)分离株A/Swine/Guangdong/9/05(H3N2)NP基因的核苷酸序列设计合成了1对带有特定酶切位点的引物,经RT-PCR扩增NP基因,将其克隆到pMD19-Simple-T载体,鉴定并测序正确后,构建pET32a-NP表达质粒,并转化大肠杆菌Rosetta(DE3),经IPTG诱导表达,SDS-PAGE电泳分析结果显示,表达的融合蛋白分子质量约为73ku,在25℃条件下主要以可溶性形式存在。将重组NP蛋白纯化后进行Western-blotting和间接免疫荧光试验,结果显示,表达蛋白可分别与H1N1、H3N2及H9N2亚型SIV阳性血清发生特异性反应,表明该蛋白具有良好的反应原性和交叉反应性;用纯化的重组NP蛋白免疫小鼠制备的多克隆血清也可与H1N1、H3N2及H9N2亚型SIV发生特异性反应,进一步证实所表达的重组NP蛋白具有良好的免疫原性。     

H5N1亚型高致病性禽流感病毒NP基因在昆虫细胞中的表达及其表达产物的生物学活性

根据GenBank上登录的H5N1亚型高致病性禽流感病毒(HPAIV)核蛋白(NP)基因序列设计引物,通过PCR扩增出A/Goose/HLJ/QFY/04(H5N1)毒株的NP基因片段,将其克隆到pFastBacGP67B杆状病毒载体上,将筛选的阳性重组转座载体pFastBacGP67B-NP转化至含有杆状病毒穿梭载体的DH10Bac感受态细胞,经蓝白斑筛选和PCR鉴定,构建了杆状病毒表达载体,获得了重组转座子(rBacmid-NP),在脂质体介导下转染对数生长期的Sf9昆虫细胞,获得重组杆状病毒(rBv-NP).SDS-PAGE分析、Westernblotting检测、免疫组化分析和ELISA试验结果表明,分子质量约为54 ku的重组核蛋白得到了高效表达,该表达蛋白具有良好的生物学活性.     

H5N1亚型禽流感病毒神经氨酸酶基因的表达及纯化

用RT-PCR方法从H5N1亚型禽流感病毒(AIV)A/Duck/Zhejiang/12/00中获得NA基因,将目的基因定向克隆到原核表达载体pET-32a中,将序列测定和双酶切验证准确的阳性重组质粒转化大肠杆菌BL21,用IPTG诱导,经SDS-PAGE和Western-blot分析,结果显示,重组蛋白得到了可溶性表达,表达的蛋白质分子质量为33 ku;该蛋白可以与禽流感H5N1亚型阳性血清反应,具有良好的免疫原性;ELISA检测结果表明,用此纯化蛋白作为包被抗原检测H5N1亚型AIV神经氨酸酶抗体具有良好的灵敏性。     

A型禽流感病毒NS1蛋白的表达及其诱导细胞凋亡功能的研究

利用瞬时表达方法表达禽流感病毒(AIV)NS1蛋白,并初步研究了体外表达的NS1蛋白对宿主细胞凋亡的影响.应用脂质体介导法,将含有不同亚型AIV NS1基因的阳性质粒pcDNA3.1(+)-qNS1(H5N1)、pcDNA3.1(+)-rNS1(H9N2)转染Vero细胞系及MDCK细胞系.经SDS-PAGE及Western-blotting检测,qNS1和rNS1基因在Vero细胞及MDCK细胞中均获得了表达.应用AO/EB荧光染色法、Annexin V-PI法分别对转染后的Vero细胞及MDCK细胞进行细胞凋亡检测,结果显示,转染阳性质粒的MDCK细胞凋亡率增高,而转染空载体pcDNA3.1(+)的MDCK细胞及转染的Vero细胞均无明显变化.表明,qNS1、rNS1蛋白在体外培养的MDCK细胞中表达并能够诱导其发生凋亡,而在Vero细胞中却不能发挥此功能.     

禽流感病毒H7亚型血凝素基因的原核表达及鉴定

参考已发表的禽流感病毒 (AIV)H7亚型血凝素 (HA)基因序列设计引物 ,经RT PCR扩增了AIV H7亚型的HA1基因片段 ,再将该片段定向插入到原核表达载体 pGEX 4T 2中 ,转化大肠埃希氏菌。重组表达载体经酶切和测序鉴定后 ,用IPTG诱导表达。用Western blotting和ELISA试验鉴定表达产物的抗原性。结果表明 ,融合表达蛋白能与AIVH7N1、H7N2、H7N7和H1N1亚型标准抗血清反应 ,而与H5N1、H9N 2等其他 13个亚型的抗血清无交叉反应。     

两株家鸭禽流感病毒H3N1亚型分离株的致病性及其表面糖蛋白基因序列分析

测定了2株禽流感病毒(AIV)Dk/YZ/231/02和Dk/YZ/3/04对SPF鸡和BALB/c小鼠的致病性；对其血凝素(HA)基因和神经氨酸酶(NA)基因的序列进行了测定,并与GenBank中收录的其他序列进行了比较。结果,此2株AIV对SPF鸡具有较低的致病性,而对BALB/c小鼠无致病性；Dk/YZ/231/02株的HA基因与Dk/Ukraine/1/63(H3N8)株的同源率最高,Dk/YZ/3/ 04株的HA基因与Petbird/HK/1559/99(H3N8)株的同源率最高；而Dk/YZ/231/02株的NA基因与Dk/Fujian/17/2001(H5N1)株的同源率最高,Dk/YZ/3/04株的NA基因与Gs/Guangdong/ 1/96(H5N1)株的同源率最高。进化分析结果表明,Dk/YZ/231/02和Dk/YZ/3/04株的NA基因均可能起源于水禽源H5N1亚型AIV,这可能是不同亚型AIV在水禽体内基因重组的结果。推导的氨基酸剪切位点序列均为PEKQTR,属于非高致病性AIV的特征序列。     

Clade2.3.4 H5亚型禽流感疫苗变异候选株的构建

为应对Clade2.3.4H5亚型禽流感病毒持续发生的抗原性改变及其引发的潜在的地方性流行,需要根据流行监测的抗原分析结果制备疫苗候选株。采用反向遗传操作系统,以A/Puerto Rico/8/34(H1N1)(PR8)AIV的内部基因为骨架,以Clade2.3.4H5N1亚型AIV A/chicken/Hebei/2/2011(wt-HB株)的血凝素(HA)基因和神经氨酸酶(NA)基因为供体,并删除wt-HB株HA基因中编码多碱性氨基酸的序列,成功拯救出1株重组病毒rHB/PR8。该病毒在鸡胚和MDCK细胞上均具有较好的繁殖性能,对SPF鸡和鸡胚无致病性。这一研究结果为防控变异的Clade2.3.4H5亚型禽流感提供了良好的疫苗候选株。     

H5亚型禽流感病毒HA1基因在Sf9昆虫细胞中的表达及其抗原性检测

利用pBlueBacHis2杆状病毒载体在昆虫细胞Sf9中表达H5亚型禽流感病毒(AIV)的 HA1基因。Western-blotting证明HA1在昆虫细胞中得到了表达,其产物具有抗原性;且不与 H7、H9 AIV的抗血清发生交叉反应,具有良好的特异性。Dot-ELISA结果显示,表达蛋白可以在非变性条件下与相应抗体作用,同样具有良好的抗原性。试验结果证明,表达的H5亚型AIV的 HA1蛋白,为检测禽类体内是否感染H5亚型AIV和监测免疫禽类血清H5抗体水平提供了优良的检测抗原。     

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.     

U.S. and East Asian Integration

the U.S. is directly impacting on the future of the East Asian community. Therefore, finding ways to get along with the U.S. is crucial to the speed, direction, configuration and character of the East Asian community. In this paper, the author has analyzed the interests of the United States in East Asia and its stands towards the East Asian integration. The author concludes that it is to the interest of the United States to make more efforts to further join in the East Asian integration. And East Asia should accept and welcome the American participation.     

CAFIU Honorary Vice-President Wang Zhizhen Visited Turkey,Sudan and Attended the Third China-Africa People's Forum

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CAFIU Delegation Visited Sri Lanka and Attended the Third China-Africa People's Forum in Sudan

正At the invitation of Sri Lanka-China Friendship Association(SLCFA)and Sudan Network for National Non-Governmental Organizations,a 5-member delegation led by CAFIU Deputy Secretary-General Liu Kaiyang visited Sri Lanka and attended the Third China-Africa People’s Forum held in Sudan from May 7 to 14,2014.As a member of this delegation,I had the great honor     

South Asian NGO Delegation Visiting China

正At the invitation of the Chinese Association for International Understanding(CAFIU),a 10-member NGO delegation composed of representatives from media,think tanks and NGOs from Bangladesh,India and Sri Lanka visited China from April 9 to 18.On the second day of arrival,the delegation visited Xicheng District Administrative Service Center.The center provides such services as     

Building A Platform for Understanding and Establishing Channels for Cooperation——Notes on the 2014 “Understanding & Cooperation” Dialogue

正From June 16 to 18,the 2014"UnderstandingCooperation"Dialogue and Forum on Rethinking Asia,co-hosted by CAFIU and FES and jointly organized with China Foundation for Peace and Development,was held in Tutzing,a beautiful and spacious manor located at 30km southwest of Munich.Nearly 60 delegates from more than 10Asian and European countries participated in the event,including China,Germany,Korea,Japan,     

The Second Sino-US Maritime Security Workshop Held in CIIS

正On September 25-26,2014,CIIS and Hoover Institution,Stanford University jointly held the Second Sino-US Maritime Security Workshop in Beijing.CIIS President Qu Xing and Abraham Sofall,senior research fellow with the Hoover Institution,codelivered the opening remarks.Representatives from China's Ministry of National Defense,     

俄白哈关税同盟的发展及其影响（英文）

In recent years, the Eurasian Economic Community has developed rapidly. Because of the special relationship between the SCO and the Customs Because Customs Union, the latter will inevitably exert some influence on the development of the former, but they are not fundamentally contradictory. Since the of the establishment of the Customs Union, the trade between China and the Customs Union members grows steadily.     

China's Development:Implications for Asian Economy

正In 2013,under the circumstance of meager recovery and continued sluggish economic growth of the whole world,China’s economy still maintained a growth rate of 7.7 per cent and the economic aggregate jumped onto a new stage-it was predicted that the share of China’s economy in the global aggregate could exceed 12 per cent compared with only 2% in 1980.The status of China as an important global economic power has been entrenched and the rise of the manufacturing     

China's Soft Power

正"Soft Power" is a concept developed by a Harvard University professor Joseph Nye in a 1990 book Bound to Lead:The Changing Nature of American Power to describe the ability to attract and co-opt rather than coerce,get others to want the outcomes you want.