| 禽流感病毒NP基因的真核表达及抗体检测芯片的建立 |
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| 引用本文： | 赵玉辉,王馥梅,包红梅,孙晓东,路冰,熊永忠,陈化兰,王秀荣.禽流感病毒NP基因的真核表达及抗体检测芯片的建立[J].中国预防兽医学报,2011,33(5). |
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| 作者姓名： | 赵玉辉 王馥梅 包红梅 孙晓东 路冰 熊永忠 陈化兰 王秀荣 |
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| 作者单位： | 1. 中国农业科学院哈尔滨兽医研究所国家禽流感参考实验室/兽医生物技术国家重点实验室,黑龙江,哈尔滨,150001
2. 浙江普洛医药科技有限公司,浙江,杭州,310000 |
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| 基金项目： | 公益性行业(农业)科研专向经费 |
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| 摘 要： | 为建立一种检测A型禽流感病毒(AIV)抗体的蛋白芯片,本研究以AIV总RNA为模板,RT-PCR方法扩增NP基因,与真核表达载体pFastBacHTa连接,并在昆虫细胞中表达.重组蛋白经纯化及western blot鉴定,点样于醛基包被的载玻片上,制备检测AIV抗体的蛋白芯片.确定芯片反应最佳条件为:点样浓度2 mg/mL,固定24 h,1%BSA进行封闭,一抗稀释度1:100,二抗稀释度1:2000.利用蛋白芯片分别对15个亚型流感病毒免疫血清、SPF鸡血清、抗新城疫病毒血清、抗法氏囊病毒血清和现地血清样品进行检测,通过与血凝抑制试验比较,表明建立的蛋白芯片方法具有良好的灵敏性和特异性,为AIV抗体检测及制备检测AIV不同亚型或多种病原抗体的蛋白芯片提供方法.
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| 关 键 词： | 流感病毒 NP蛋白 真核表达 蛋白芯片 |
Preparation of protein microarray analysis with eukaryotic expressed NP protein for detection of avian influenza virus antibody |
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| ZHAO Yu-hui,WANG Fu-mei,BAO Hong-mei,SUN xiao-dong,LU Bing,XIONG Yong-zhong,CHEN Hua-lan,WANG Xiu-rong.Preparation of protein microarray analysis with eukaryotic expressed NP protein for detection of avian influenza virus antibody[J].Chinese Journal of Preventive Veterinary Medicine,2011,33(5). |
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| Authors: | ZHAO Yu-hui WANG Fu-mei BAO Hong-mei SUN xiao-dong LU Bing XIONG Yong-zhong CHEN Hua-lan WANG Xiu-rong |
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| Institution: | ZHAO Yu-hui1,WANG Fu-mei2,BAO Hong-mei1,SUN xiao-dong1,LU Bing1,XIONG Yong-zhong1,CHEN Hua-lan1,WANG Xiu-rong1 (1. State Avian Influenza Reference Laboratory and State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China,2. Zhejiang Apeloa Jiayuan Pharmaceutical Co. Ltd,Hangzhou 310000,China) |
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| Abstract: | This paper described a protein microarray, which could be used into detect antibody of influenza A virus (AIV) using recombinant NP protein. The NP gene was amplified by RT-PCR from viral RNA and cloned into the eukaryotic expression vector pFastBacHTa to express recombinant protein. The recombinant protein was identified by western blot,purified through Ni+ affinity chromatography and spotted on the aldehyde-coated slides to prepare protein microarray. The optimized conditions of the assay were included a ... |
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| Keywords: | influenza virus NP protein eukaryotic expression protein microarray |
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