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2012年27卷2期

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Research Article

杆状病毒表达载体的遗传改造*

李淑芬, 胡志红, 邓菲,

2012, 27(2): 71 doi: 10.1007/s12250-012-3236-y

[HTML全文] [PDF 390 KB] Springerlink

杆状病毒作为蛋白表达载体,与其他载体相比有着许多优势。随着生物技术的不断发展,我们可以通过对杆状病毒的遗传改良,来促进外源蛋白在昆虫和哺乳动物细胞中的大量表达。这些改造包括:运用不同的启动子和信号肽,缺失或者替换某些病毒的基因来提高分泌型蛋白的表达量;在基因组中整合多顺反子表达框来表达蛋白复合体;以及通过对启动子的改造和表面展示等方法来达到外源基因对于哺乳动物细胞的定向转移,进而用于基因治疗等。杆状病毒在蛋白表达和基因治疗方面有着很大的潜力,对于杆状病毒表达系统的进一步优化将使它在未来得到更多更广的应用。

内源性绵羊肺腺瘤病毒及其受体Hyal-2 mRNA在绵羊胎儿和新生羔羊免疫器官中的表达

齐景伟, 吴晓利, 刘淑英, 曹贵方

2012, 27(2): 83 doi: 10.1007/s12250-012-3222-4

[HTML全文] [PDF 892 KB] Springerlink

内源性绵羊肺腺瘤病毒与引起绵羊肺腺瘤病的病原绵羊肺腺瘤病毒非常相似,为了揭示感染绵羊肺腺瘤病的病羊血清中检测不到循环抗体的免疫学机理,本研究采用Real-Time 荧光定量PCR和原位杂交技术检测内源性绵羊肺腺瘤病毒(enJSRV)及其受体Hyal-2 mRNA在绵羊胎儿和羔羊的免疫器官和肺脏中的表达情况。原位杂交结果表明enJSRV和 Hyal-2 mRNA在不同时期绵羊的胸腺、脾脏、肠系膜淋巴结和肺脏中均有表达,而阴性对照组均没有阳性信号出现。Real-Time 荧光定量PCR检测结果表明enJSRV Env 在130d的胎儿和3日龄羔羊的免疫器官中的mRNA表达水平明显高于在肺脏中的表达,特别是在胸腺和脾脏中表达更高。但是受体Hyal-2 在这些组织中的表达水平无明显差异。这些结果从免疫学的角度为感染绵羊肺腺瘤病毒的病羊体内检测不到循环抗体的事实提供了证据,同时也为理解该病的发病机理提供理论数据。

Characterization of Pigeon Paramyxoviruses (Newcastle disease virus) Isolated in Kazakhstan in 2005

Andrey Bogoyavlenskiy, Vladimir Berezin, Alexey Prilipov, Eugeniy Usachev, Ilya Korotetskiy, Irina Zaitceva, Aydyn Kydyrmanov, Marat Sayatov

2012, 27(2): 93 doi: 10.1007/s12250-012-3234-0

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Isolates of Newcastle disease virus (NDV) from deceased wild and domestic pigeons in Kazakhstan were obtained from the Almaty region during 2005 and were genotypically analyzed by using reverse transcription polymerase chain reaction (RT-PCR) with primers specific to the viral fusion (F) protein gene. Part of the amplified F protein DNA product (nucleotide sequence 47–422) and the deduced amino acid sequences were compared phylogenetically with those from strains previously reported in other geographic regions. Phylogenetic analysis indicated that the Kazakhstanian pigeon paramyxovirus type 1 (PPMV-1) isolates belong to genotype VI or 4bii. To our knowledge, this is the first reported VI isolates that possess the sequences of 112 GKRQKR116* F117 within the F0 protein. The information is fundamental to improving the efficiency of control strategies and vaccine development for NDV.

水稻条纹病毒NSvc2蛋白细胞膜表面展示与膜融合分析

赵淑玲, 戴雪娟, 梁建生, 梁昌镛

2012, 27(2): 100 doi: 10.1007/s12250-012-3237-x

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水稻条纹病毒(RSV)感染水稻,通过介体灰飞虱以循环增值的方式传播。目前对于RSV如何进入昆虫细胞并且起始感染的机制了解的很少。序列分析显示,RSV NSvc2与布尼亚病毒科一些病毒的膜蛋白有相似性,暗示它可能具有诱导膜融合的功能。为了更便捷研究NSvc2的膜融合活性,本文构建了昆虫细胞表面展示系统。结果显示,利用该系统RSV NSvc2成功在昆虫细胞内表达并被转运到细胞膜表面。低pH诱导表达有NSvc2的细胞并观察是否有合胞体的形成,结果表明,低pH条件下NSvc2没有诱导膜融合。另外,当NSvc2与病毒的衣壳蛋白(CP)在昆虫细胞内共表达时,也没有观察到膜融合产生。因此,NSvc2可能与其同源性的膜蛋白不同,可能具有不同的功能。RSV可能不是通过与细胞膜或内吞体膜融合的方式进入昆虫细胞的。

草鱼呼肠孤病毒(GCRV)体外抑制RNA干扰途径(RNAi)

郭帅, 许丹, 徐鸿绪, 王土, 李家乐, 吕利群

2012, 27(2): 109 doi: 10.1007/s12250-012-3230-4

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草鱼呼肠孤病毒(GCRV)基因组双链RNA(dsRNA)逃逸双链RNA触发式(dsRNA-triggered)和Dicer蛋白启动式(Dicer-initiated)的RNA干扰机制(RNAi)的方法,目前有待于进一步的研究。本研究的目的在于探讨草鱼肾细胞(CIK)中RNA干扰机制对草鱼呼肠孤病毒复制的影响。通过对RNA干扰机制影响的分析,结果表明,草鱼肾细胞中存在完整的双链RNA触发式RNA干扰机制。通过将GCRV特异性小干扰RNA(siRNA)与纯化的GCRV基因组双链RNA与草鱼肾细胞共转染,并用Northern印迹法(Northern Blot)检测分析,草鱼呼肠孤病毒感染细胞中,并未检测到GCRV特异的siRNA。该实验进一步证明,草鱼呼肠孤病毒的复制与Dicer蛋白基因转录水平的增加有关,在此过程中,抑制了人工合成的EGFP特异的siRNA沉默EGFP基因。这些数据证明,病毒基因组的双链RNA对RNA干扰机制是敏感的,但是未知的RNA干扰抑制蛋白(组)可能有助于病毒基因组的存活和病毒复制高效率。

动物流感病毒通用多重RT-PCR扩增及液相芯片高能量分型检测*

秦智锋, 孙洁, 卢体康, 曾少灵, 花群义, 林庆燕, 陈书琨, 吕建强, 张彩虹, 陈兵, 阮周曦, 毕英佐, Joseph J Giambrone, 吴红专

2012, 27(2): 120 doi: 10.1007/s12250-012-3232-2

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为了对流感病毒阳性毒株进行快速分型鉴定,建立了新型GM RT-PCR扩增检测技术,通过引入通用超级引物来实现一次PCR扩增实现多个目标片段同时扩增的目的,再与液相芯片高通量检测技术相结合,建立了流感GMPLex快速高通量分型检测方法,达到对流感病毒快速、准确、高通量分型检测的目的。首先设计了针对流感病毒的NS基因、流感病毒的M基因、流感病毒常见亚型H1亚型、H3亚型、H5亚型、H7亚型、H9亚型的HA基因和N1亚型、N2亚型的NA基因的特异性分型引物和探针,开创性地引入了通用超级引物,创建了包括富集、加尾、PCR扩增三个阶段的全新通用多重RT-PCR(GM RT-PCR)检测方法,实现了对流感病毒多个亚型的目标片段一次多重RT-PCR扩增目的,解决了芯片检测过程中需要多次PCR扩增的“瓶颈”。将流感病毒分型GM RT-PCR检测方法与液相芯片高通量检测方法结合,建立了流感病毒GMPLex快速高通量分型检测方法。所建流感GMPLex快速高通量分型检测方法检测通量高,可一次对A型流感病毒的9个目的基因7个流感亚型包括H1亚型、H3亚型、H5亚型、H7亚型、H9亚型和N1亚型、N2亚型进行分型鉴定;快速,流感分型检测可在6h内完成;特异性强,通过两套套式简并引物和一个特异性探针来确保每个亚型的特异性,所建立的多亚型GMPlex快速高通量检测方法检测流感各个亚型毒株,相互之间没有发现交叉反应,与其他病原体之间也无非特异性反应;灵敏度高,多亚型GMPlex快速高通量检测方法检测H5基因单亚型的检测灵敏度为病毒尿囊液稀释至10-5(相当于280 ELD50),检测N1基因单亚型的检测灵敏度为病毒尿囊液稀释至10-5(相当于280 ELD50)。多亚型GMPlex快速高通量检测方法检测H5N1单毒株检测灵敏度为病毒尿囊液稀释至10-4(相当于2800 ELD50),可同时鉴定出H5亚型和N1亚型。采用所建立的GMPLex快速高通量分型检测方法对实验室保存的83株禽病病毒株进行小样本检测,检测结果与经典检测方法结果一致,证明所建立的GMPLex快速高通量分型检测方法能够满足口岸禽流感病毒检测快速高通量的要求,也搭建了全新的禽流感病毒快速高通量检测平台。

Analysis on Factors Related to Rabies Epidemic in China from 2007-2011*

Cui-ping Yin, Hang Zhou, Hui Wu, Xiao-yan Tao, Simon Rayner, Shu-mei Wang,

2012, 27(2): 132 doi: 10.1007/s12250-012-3244-y

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To analyze features of the rabies epidemic in China between 2007 and 2011, identify factors influencing the epidemic and to provide a scientific basis for further control and prevention of rabies, Descriptive epidemiological methods and statistical analysis was used on data collected from the National Disease Reporting Information System between 2007 to 2011 and the National Active Surveillance System between 2007 and 2010. Our analysis shows that while the number of human rabies cases decreased year by year, the number of districts reporting cases did not show significant change. The situations in Guangdong, Guangxi, Guizhou and Hunan provinces clearly improved over the period but they remain provinces with high-incidence, and consequently influence the epidemic situation of surrounding provinces and possibly the whole country. Summer and autumn were high-incidence seasons. Farmers, students and pre-school children represent the high-risk populations, and rates of cases in farmers increased, those for students decreased, and pre-school children remained unchanged. Provinces with active surveillance programs reported a total of 2346 individual cases, of which 88.53% were associated with canines. Postexposure prophylaxis (PEP) of rabies cases was not significantly improved, whereas PEP in post-exposure population was good. In rural regions of China, canine density was reduced somewhat, and the immunization rate increased slightly. Finally we show that while the epidemic decreased 2007 to 2011 in China, cases continued to be diffused in certain regions. Lack of standardization of PEP on rabies cases was the main reason of morbidity. The high density and low immunization of dog in rural areas and the defective situation of PEP are still continuous occurrences in China and remain a cause for concern.