| 鸭肠炎病毒gD基因胞外区的原核表达及在感染鸭胚成纤维细胞中的定位 |
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| 引用本文： | 刘琰,徐凝,付培芬,母晓宇,董井泉,马波,王君伟.鸭肠炎病毒gD基因胞外区的原核表达及在感染鸭胚成纤维细胞中的定位[J].畜牧兽医学报,2012,43(6):922-927. |
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| 作者姓名： | 刘琰 徐凝 付培芬 母晓宇 董井泉 马波 王君伟 |
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| 作者单位： | 东北农业大学动物医学学院,哈尔滨,150030 |
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| 基金项目： | 高等学校博士学科点专项科研基金资助课题，黑龙江省科技攻关项目，东北农业大学博士启动基金 |
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| 摘 要： | 以本实验室获得的鸭肠炎病毒(DEV)C-KCE株US区基因序列(GenBank登录号EU714267)为基础PCR扩增出gD基因胞外区(gDw),将其定向克隆至原核表达载体pET-30a(+)并转化至大肠杆菌RosettaTM(DE3)pLys。经IPTG诱导表达,SDS-PAGE分析表明gD基因胞外区在大肠杆菌中获得与预期大小相符的表达蛋白。重组蛋白纯化后免疫家兔制备多克隆抗体,血清琼扩效价达1∶16,噬斑减数中和试验测定血清中和抗体效价达1∶64。通过免疫荧光技术首次对gD进行亚细胞定位检测,结果表明DEV感染鸭胚成纤维细胞(DEF)后4h近核区域细胞质内首先开始出现荧光,12h以后显著增加,明显的点状绿色荧光广泛存在于胞质中。本研究为进一步开展DEV gD的功能研究提供了重要数据和材料。
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| 关 键 词： | 鸭肠炎病毒 gD 原核表达 噬斑减数中和试验 亚细胞定位 |
Prokaryotic Expression of DEV Extracellular gD Gene and Its Subcellular Localization in Virus-infected DEF |
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| LIU Yan
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XU Ning
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FU Pei-fen
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MU Xiao-yu
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DONG Jing-quan
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MA Bo
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WANG Jun-wei.Prokaryotic Expression of DEV Extracellular gD Gene and Its Subcellular Localization in Virus-infected DEF[J].Acta Veterinaria et Zootechnica Sinica,2012,43(6):922-927. |
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| Authors: | LIU Yan XU Ning FU Pei-fen MU Xiao-yu DONG Jing-quan MA Bo WANG Jun-wei |
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| Institution: | 1* (College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China) |
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| Abstract: | The extracellular gD gene was amplified by PCR from DEV genomic DNA according to our laboratory discovered gene sequence(GenBank accession number EU714267).The recombinant expression plasmid pET-30a-gDw could effectively express gDw fusion protein that was used to immunize rabbits.The titer of antibody tested by agar diffusion reaction was 1:16.Plaque reduction neutralization test showed that its neutralization antibody titer was up to 1:64.By indirected immunofluorescence,the results showed that gD appeared in the cell cytoplasm near the nuclear as early as 4 hours post infection.After 12 hours post infection,the fluorescence in cytoplasm was increased.Above all,the results afforded significant data for the study on the function of DEV gD. |
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| Keywords: | duck enteritis virus gD prokaryotic expression plaque reduction neutralization test subcellular localization |
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