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首页> 外文期刊>The Kasetsart Journal >Cloning and in vitro Expression of N1 Neuraminidase Gene of Avian Influenza Virus A/Duck/Thailand/KU-KPS/2004(H5N1)
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Cloning and in vitro Expression of N1 Neuraminidase Gene of Avian Influenza Virus A/Duck/Thailand/KU-KPS/2004(H5N1)

机译：禽流感病毒A /鸭/泰国/ KU-KPS / 2004（H5N1）N1神经氨酸酶基因的克隆和体外表达

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摘要

Avian influenza virus (AIV) can spread rapidly and causes serious disease to animals and humans so it is crucial to have effective diagnostic tools for determining its existence. The purposes of this study were first, to clone N1 neuraminidase (NA) gene of AIV A/Duck/Thailand/KU-KPS/2004 (H5N1), and second, to express recombinant N1 using baculovirus expression system. Viral RNA extracted from positive AIV allantoic fluid was reverse-transcribed to cDNA and then was amplified by PCR using N1 specific primers. The PCR products approximately 1,300 bp that encode the N1 was introduced into baculovirus vector in order to allow N1 expression in insect cell lines. Immuno-dot blot analysis of crude extract of baculovirus infected insect cells using goat-anti H5N1 AIV hyperimmune serum gave a clear immunoreactive spot. SDS-PAGE analysis showed banding patterns from the crude extract contained major protein of 54 kDa. Western blot analysis using goat-anti H5N1 hyperimmune serum and mouse-antihistidine monoclonal antibody also gave a specific band approximately the same size as that of SDS- PAGE. Expression analysis of this gene indicated that recombinant neuraminidase may have correct post-translation modification and folding. The recombinant neuraminidase could be very useful for the development of a test kit that can differentiate infected from vaccinated animals (DIVA).
机译：禽流感病毒（AIV）可以迅速传播并对动物和人类造成严重的疾病，因此拥有有效的诊断工具来确定其存在至关重要。本研究的目的是首先克隆AIV A / Duck / Thailand / KU-KPS / 2004（H5N1）的N1神经氨酸酶（NA）基因，其次，使用杆状病毒表达系统表达重组N1。从阳性AIV尿囊液中提取的病毒RNA反转录为cDNA，然后使用N1特异性引物通过PCR进行扩增。为了允许N1在昆虫细胞系中表达，将编码N1的大约1300bp的PCR产物引入杆状病毒载体中。使用山羊抗H5N1 AIV超免疫血清对杆状病毒感染的昆虫细胞粗提液进行免疫斑点印迹分析，得到了清晰的免疫反应点。 SDS-PAGE分析显示，粗提物的条带模式包含54 kDa的主要蛋白质。使用山羊抗H5N1超免疫血清和小鼠抗组氨酸单克隆抗体的Western印迹分析还产生了与SDS-PAGE大小大致相同的特异性条带。该基因的表达分析表明重组神经氨酸酶可能具有正确的翻译后修饰和折叠。重组神经氨酸酶对于开发可区分感染动物和疫苗接种动物（DIVA）的测试试剂盒可能非常有用。

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