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| 传染性造血器官坏死病毒糖蛋白抗血清的制备及应用 |
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朱旭1,2,3, 兰文升2,3, 刘荭2,3, 高隆英2,3, 杜鹃1,2,3, 陈孝煊1
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1.华中农业大学水产学院，武汉 430070;2.深圳出入境检验检疫局动植物检验检疫技术中心，518045;3.深圳市外来有害生物检测技术研发重点实验室，518045
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| 摘要: |
| 应用逆转录聚合酶链式反应（RT-PCR）从传染性造血器官坏死病毒（Infectious hematopoietic necrosis virus，IHNV）感染的细胞悬液克隆病毒的糖蛋白基因，将其亚克隆至原核表达载体pCWori，转化到大肠杆菌DH5α，通过发酵大肠杆菌制备病毒糖蛋白。经SDS-PAGE分析，诱导表达的重组蛋白主要以包涵体的形式存在，使用Ni-NTA亲和层析柱在变性条件下进行纯化并透析复性，最终得到了较高纯度的可溶性糖蛋白，分子量约为57 kDa。Western-blot分析结果显示，所表达的蛋白能够被IHNV病毒制备的兔抗IHNV血清识别。用复性后的蛋白免疫小鼠制备抗血清， ELISA显示抗体效价可达1∶64 000。经制备的抗血清可以作为一抗建立ELISA检测方法，用于检测细胞悬液的病毒粒子，将抗血清稀释到1∶16 000仍能与IHNV全病毒发生反应。本研究利用重组的IHNV糖蛋白成功制备了高效价的抗血清，并能够与IHNV全病毒发生特异性结合，为IHNV免疫学检测方法建立奠定了基础。 |
| 关键词: 传染性造血器官坏死症病毒 糖蛋白 原核表达 抗血清 |
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| 基金项目:国家质检公益性行业科研专项项目（201010020） |
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| Antiserum preparation for infectious hematopoietic necrosis virus (IHNV) glycoprotein and its application in IHNV detection |
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| Abstract: |
| The 1527bp IHNV glycoprotein gene was cloned by reverse transcription polymerase chain reaction（RT-PCR）from cell culture, and was subcloned into pCWori plasmid. Then the recombinant pCWori-G was transformed into DH5α. The recombinant protein was obtained through fermentation. The 57kDa target protein was expressed successfully by 1 mmol/L IPTG induction for 5 h. SDS-PAGE analysis showed that it was mainly in the form of inclusion body. The soluble glycoprotein was obtained after purification and renaturation processing. Western blot analysis showed that the recombinant protein was identified specifically by the goat anti IHNV serum. The recombinant protein was used to immunize mice to produce glycoprotein antiserum. The prepared antiserum reacted specifically with IHNV by indirect ELISA test. In this study, the IHNV glycoprotein was expressed by prokaryotic expression system and polyclonal antiserum was obtained by immunized mice. |
| Key words: Infectious hematopoietic necrosis virus Glycoprotein Prokaryotic expression Antiserum |