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2010年25卷6期

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Research Article

Detection of Bluetongue Virus Group-specific Antigen Using Monoclonal Antibody Based Sandwich ELISA

Pradeep Narayan, Veerakyathappa Bhanuprakash, Vinayagamurthy Balamurugan, Madhusudhan Hosamani, Gnanavel Venkatesan, and Raj

2010, 25(6): 390 doi: 10.1007/s12250-010-3160-y

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A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA. The MAb, designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study. The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein. The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits. The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India, 1, 2, 15, 17, 18 and 23. The assay could detect BTV antigen as early as day 8 in blood. It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood, washed RBCs, buffy coat and plasma. A total of 102 field samples from animals, suspected of being infected with BTV, were tested and 29.42% were positive. The blood samples were also amplified in cell culture which improved the sensitivity of the assay. Results confirmed that the sELISA is rapid and specific.

水稻瘤矮病毒外层衣壳蛋白基因S8在昆虫细胞中的表达?

cheng FAN, luan GAO, yun WEI, ying HUANG, yan XIE, jian WU, ying LIN, hui XIE

2010, 25(6): 401 doi: 10.1007/s12250-010-3152-y

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为了获得具有生物活性的水稻瘤矮病毒(Rice gall dwarf virus, RGDV)外层衣壳蛋白P8,利用杆状病毒表达系统对其主要外层衣壳蛋白基因S8在草地贪夜蛾(Spodoptera frugiperda, Sf9)昆虫细胞中进行表达。将RGDV外层衣壳蛋白基因S8亚克隆至杆状病毒表达载体pFastBacTM1上,获得重组杆状病毒转移载体pFB-S8,以pFB-S8转化含穿梭质粒Bacmid的E. coli DH10Bac感受态细胞,得到含有目的基因片段的重组杆粒rbpFB-S8。重组病毒rvpFB-S8以不同MOI(Multiplicity of infection, MOI)感染Sf9细胞,不同时间段收集细胞,并进行SDS-PAGE,Western-blotting分析以及免疫荧光显微镜观察。结果表明,S8 基因在昆虫细胞中成功表达,在感染Sf9昆虫细胞48-72 h后RGDV P8蛋白表达量达最高,免疫荧光显微镜观察则显示,RGDV P8蛋白在感染的Sf9昆虫细胞质中形成虚线状结构。

Sequence Analysis of Attachment Gene of Lumpy Skin Disease and Sheep Poxviruses

A. A. El-Kenawy, M. S. El-Tholoth

2010, 25(6): 409 doi: 10.1007/s12250-010-3150-0

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Egypt, protection of cattle against lumpy skin disease (LSD) was carried out using a sheep poxvirus (Kenyan strain) vaccination strategy. In the present study 15 skin nodules from LSD suspected cows and 5 scab samples from sheep pox (SP) suspected sheep were collected. Hyperimmune rabbit sera to Lumpy skin disease virus (LSDV)/Ismailyia88 strain and sheep pox virus (SPV)/ Kenyan vaccinal strain were prepared. The causative agent in the collected samples was identified using immunoflourescence (IF) and immunoperoxidase techniques. Of the 15 skin nodules suspected of LSD, 10 showed a positive reaction and 3 out of 5 skin scabs suspected of sheeppox were found to be positive. An antigenic correlation between field skin isolate of LSDV, tissue culture adapted LSDV/Ismailyia88 strain, field skin isolate of SPV and SPV/Kenyan vaccinal strain was studied using prepared hyperimmune sera. Also, nucleotide sequence of the PCR amplified attachment gene fragments of field skin isolate of LSDV, tissue culture adapted LSDV/Ismailyia88 strain, field skin isolate of SPV and SPV /Kenyan vaccinal strain were compared. The results revealed that the four used viruses were antigenically identical. Sequence analysis indicated that field skin LSDV isolate is more related to tissue culture adapted LSDV/Ismailyia88 strain than to vaccinal SPV/ Kenyan strain and the skin isolate of SPV is more closely related to field skin isolate of LSDV than to SPV/Kenyan vaccinal strain. Thus, further study should be applied on the advantage of a LSD vaccine prepared from LSDV in protection of cattle against LSD compared to the commonly used sheep pox vaccine.

HSV1感染相关细胞分子HTRP通过乙酰化体系对病毒转录调控的影响?

Jie CHEN, Yan-mei LI, Jian-feng LI, Long-ding LIU, Yun LIAO, Rui-xiong NA, Jing-jing WANG, Li-chun WANG, Qi-han LI*

2010, 25(6): 417 doi: 10.1007/s12250-010-3147-8

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Ⅰ型单纯疱疹病毒(HSV-1)感染KMB-17后诱导的差异基因htrp所编码蛋白HTRP可与去乙酰化转移酶HDAC辅抑制因子复合物mSin3A的组成成分之一SAP30之间发生相互作用。为了进一步揭示HTRP与SAP30相互作用的生物学意义,本实验利用实时荧光定量PCR及双荧光素酶检测系统,证明HTRP对病毒启动子的转录具有抑制作用,其和SAP30共同作用对病毒基因的转录抑制具有协同效应,且这种效应与HDAC的酶活性相关。ChIP实验证明其具体机理是HTRP能够促进HDACs酶活性而增加组蛋白H3分子第14和9位赖氨酸的去乙酰化水平。

H5N1感染鸡胚成纤维细胞中标化定量PCR结果的参考基因选择

Hua YUE, Xiao-wen LEI, Fa-long YANG, Ming-Yi LI, Cheng TANG

2010, 25(6): 425 doi: 10.1007/s12250-010-3114-4

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鸡胚成纤维细胞(CEF)是研究H5N1禽流感病毒与宿主之间相互作用时最常用的细胞之一。本研究对H5N1病毒感染CEF细胞后11个持家基因mRNA表达的稳定性进行了比较,旨在获得定量PCR研究中对数据进行标化的可靠内参基因。试验采用100TCID50 H5N1病毒感染CEF,在感染后3、12、24、30小时收获细胞,采用定量PCR技术和GeNorm工具软件,对11个持家基因的表达水平和稳定性进行比较研究。结果表明:在正常CEF和病毒感染的CEF细胞中,11个持家基因表达稳定性不同; RPL4、YWHAZ是研究H5N1病毒感染后宿主细胞基因表达的理想内参基因。对于H5N1禽流感病毒在CEF中复制的相关研究,ACTB以及RPL4则是理想的内参基因。

重组蓝藻抗病毒蛋白-N抗HSV-1活性的研究*

Hong YU, tao LIU, Rui LV, qing ZHANG

2010, 25(6): 432 doi: 10.1007/s12250-010-3131-3

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探讨重组蓝藻抗病毒蛋白N 体内外抗单纯疱疹病毒1型(HSV-1) 的作用。选用HSV-1标准株(SM44株)进行了CV-N抗病毒活性的研究。 通过观察细胞病变效应(CPE)、MTT比色法检测细胞增殖判断CV-N在Vero细胞中的抗病毒效果;荧光定量PCR(FQ-PCR)检测病毒DNA的拷贝数。结果显示CV-N对Vero细胞毒性较低,半数毒性浓度CC50为359μg/ml;CV-N对HSV-1无直接灭活作用,在病毒感染前及感染后应用CV-N均可有效地抑制病毒的复制,其IC50分别是2.26 和30.16μg/mL,CV-N还可以明显抑制HSV-DNA的的复制。体内实验通过昆明小鼠脑内局部接种HSV-1,制备疱疹性脑炎模型,分别于病毒接种后2h、3d、5d、7d 以3个不同剂量的的CV-N( 0.5, 5, 10 mg/kg) 腹腔给药治疗,观察记录小鼠的发病时间及存活时间,同时取脑组织标本进行HE染色观察细胞病变。结果表明,与病毒对照组相比,5mg/kg及10mg/kg CV-N治疗组小鼠症状明显减轻,平均存活天数分别超过9天和14天,HE染色显示脑组织仅有轻微的炎症改变。本研究表明CV-N在体内、外均具有良好的抗病毒活性,是治疗HSV感染的潜力药物之一 。

朊蛋白多肽PrP106-126 改变了小鼠小胶质细胞BV-2的 PrP mRNA表达

Yu BAI, rong LI, hua WANG, mei ZHOU, ming ZHAO

2010, 25(6): 440 doi: 10.1007/s12250-010-3134-z

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朊病是一种传染性和致死性的神经退行性疾病。朊病病原是一种异常的朊蛋白聚集物。中枢神经系统内的小胶质细胞激活是朊病的一种显著特征。本研究应用实时荧光定量PCR方法检测了朊蛋白多肽PrP106-126对小鼠小胶质细胞BV-2 的PrP mRNA表达影响。结果显示PrP106-126作用BV-2细胞18 h 后PrP mRNA表达水平显著提高,比正常水平提高3倍,作用24 h 后比正常水平提高4.5 倍,且BV-2 细胞的增殖活性在18 h 和24 h 后也相应提高。这些结果首次证明了朊蛋白多肽PrP106-126 提高了小胶质细胞的PrP mRNA 表达水平,表明小胶质细胞在朊病毒致病过程中可能具有关键作用。

大青叶有效单体体内抗流感病毒活性的研究

Zhao LIU, Zhan-qiu YANG, Hong XIAO

2010, 25(6): 445 doi: 10.1007/s12250-010-3142-0

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为评价大青叶有效单体在体内抗流感病毒活性,我们建立了病毒性肺炎小鼠模型,分为3个不同剂量(低,中,高)组,然后观察它们的肺指数,肺病变,肺病毒滴度,存活时间和死亡率。结果表明:低,中,高剂量的大青叶单体可降低小鼠肺指数从2.64至1.93,1.63和1.40(P <0.01),病毒血凝滴度可从1.15下降至0.84,0.70和0.59(P <0.01)。此外,不同剂量的大青叶均能将小鼠死亡率由100%降为30%,25%和15%,延长小鼠生存时间从5.1d至6.5d,8.4d和8.9d(P<0.01)。高剂量组(75 mg.kg-1.d-1)与100 mg.kg-1/d病毒唑同效(P> 0.05),优于200 mg.kg-1.d-1抗病毒口服液性(P <0.05)。