[1]葛 叶,邓国华*,李雪平,等.流感病毒聚合酶PA亚单位抗血清的制备[J].中国预防兽医学报,2009,(04):314-317.
 GE Ye,DENG Guo-hua*,LI Xue-ping,et al.Preparation of polyclonal antibody of avian influenza virus polymerase PA subunit[J].Chinese journal of preventive veterinary medicine,2009,(04):314-317.
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流感病毒聚合酶PA亚单位抗血清的制备
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2009年04期
页码:
314-317
栏目:
免疫学
出版日期:
2009-04-15

文章信息/Info

Title:
Preparation of polyclonal antibody of avian influenza virus polymerase PA subunit
作者:
葛 叶邓国华*李雪平高玉伟姚秋成唐艳玲陈化兰
(中国农业科学院哈尔滨兽医研究所 农业部动物流感重点开放实验室/兽医生物技术国家重点实验室,黑龙江 哈尔滨 150001)
Author(s):
GE Ye DENG Guo-hua* LI Xue-ping GAO Yu-wei YAO Qiu-cheng TANG Yan-ling CHEN Hua-lan
(Animal Influenza Laboratory of Ministry of Agriculture/State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China)
关键词:
禽流感病毒PA基因抗血清
Keywords:
avian influenza virus PA gene anti-serum
文献标志码:
A
摘要:
本研究采用RT-PCR方法从H5N1亚型禽流感病毒(AIV) A/Duck/Zhejiang/11/00中分段及全长扩增PA基因片段,将目的基因定向克隆至原核表达载体pET-32a,经测序验证正确后,获得重组阳性质粒,将重组质粒转化BL21细菌后诱导,实现质粒在大肠杆菌中的表达,重组表达蛋白经Ni+柱纯化后分别与弗氏免疫佐剂乳化成油乳剂苗免疫新西兰大耳白兔,获得抗PA的免疫抗血清。Western blot检测结果表明抗血清与目的蛋白发生特异性反应。同时,将PA全长基因插入真核表达载体pCAGGS,转染293T细胞,通过激光共聚焦显微镜发现,抗血清与PA基因的真核表达产物发生反应,出现特异性荧光,在细胞核与细胞浆中均出现荧光,进一步表明PA亚单位在细胞核与细胞浆均有分布。
Abstract:
In this study, the segments and full-length PA fragment of avian influenza virus (AIV) subtype H5N1 were amplified by RT-PCR from the cDNA of AIV subtype H5N1 strain A/Duck/Zhejiang/11/00, cloned into pET-32a vector and expressed in E.coli. The expressed protein was purified by Ni+ column and used to immuize rabbit. Antibody specifically to the fusion protein was obtained and confirmed by western blot. The full-length PA gene was inserted into eukaryotic expression pCAGGS vector and transfected into 293T cells. Expression of protein was detected by fluorescence labeled anti-serum. The results showed that the anti-serum could react with nature and recombinant PA protein distributed throughout the cytoplasm of the cell.

参考文献/References:

[1] Helena J Maier, Takahito Kashiwagi, Koyu Hara ,et al. Differential role of the influenza A virus polymerase PA subunit for vRNA and cRNA promoter binding [J].Virology, 2008, 370(1): 194-204.

[2] Simon M Carr, Elena Carnero, Adolfo Garc?´a-Sastre, et al. Characterization of a mitochondrial-targeting signal in the PB2 protein of influenza viruses [J].Virology, 2006, 344(2): 492-508.

[3] Koyu Hara, Florian I Schmidt, Mandy Crow, et al. Amino Acid Residues in the N-Terminal Region of the PA Subunit of Influenza A Virus RNA Polymerase Play a Critical Role in Protein Stability, Endonuclease Activity, Cap Binding, and Virion RNA Promoter Binding [J].J Virol, 2006, 80(16): 7789–7798.

[4] 萨姆布鲁克J,拉塞尔D W. 分子克隆实验指南[M] . 黄培堂,王嘉玺,朱厚础,等译. 3. 北京:科学出版社, 2002.

[5] 薛庆善主编.体外培养的原理与技术[M]. 北京:科学出版社,2001.

[6] Fodor E, Smith MThe PA subunit is required for efficient nuclear accumulation of the PBl subunit of the influenza A virus RNA polymerase complex [J]. J Virol200478(17)9144-9153

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备注/Memo

备注/Memo:
收稿日期:2008-10-07
基金项目:国家十五科技攻关项目(2004BA519A54)
作者简介:葛 叶(1982-),女,黑龙江伊春人,硕士研究生,主要从事禽流感病毒分子生物学基础研究.
*通信作者:E-mail:dgh1971@163.com
更新日期/Last Update: 2009-06-09