番鸭呼肠孤病毒σA蛋白的原核表达及其多克隆抗体的制备  

Prokaryotic expression of σA protein of Muscovy duck reovirus and preparation of polyclonal antibody

作　　者：吕小婷[1] 廖加磊 孙雪宁[1] 胡万荣[1] 朱二鹏 吴异健[1] 吴宝成[1] 

机构地区：[1]福建农林大学动物科学学院,福建福州350002

出　　处：《中国兽医科学》2016年第2期174-179,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(U1305212);福建省自然科学基金项目(2012J01067)

摘　　要：为实现番鸭呼肠孤病毒（MDRV）σA蛋白的原核表达及其多克隆抗体的制备,采用RT-PCR扩增了MDRV-YB株σA基因的完整编码序列（CDS）,双酶切并连接至表达载体p ET-32a（＋）中,将重组表达质粒转化大肠杆菌BL21（DE3）,IPTG诱导蛋白表达,并对表达产物进行SDS-PAGE分析;随后对重组σA蛋白进行Ni2＋柱亲和层析纯化,用Western-blot鉴定纯化后重组蛋白的活性;最后以纯化后的包涵体蛋白为抗原免疫新西兰兔,制备多克隆抗体,并用ELISA法检测其效价,Western-blot鉴定其特异性。SDS-PAGE分析表明,成功表达出分子质量约为64.2 ku的融合蛋白,且主要以包涵体形式存在;其IPTG诱导最佳时间、浓度和温度分别为6 h、1.2 mmol/L和24~27℃;经Ni2＋柱纯化获得了高纯度重组σA蛋白;Western-blot结果显示,纯化前、后的重组蛋白均可以与MDRV阳性血清发生特异性识别,说明纯化后的重组σA蛋白依然保留良好的反应原性;ELISA测得多抗血清的效价大于1∶32 000,Western-blot鉴定进一步证实制备的多克隆抗体可与重组σA蛋白发生特异性反应,为MDRVσA蛋白生物学功能研究奠定了基础。To express and prepare a polyclonal antibody against σA protein of Muscovy duck reovirus（MDRV）,the σA gene of MDRV-YB strain was amplified by RT-PCR and cloned into the expression plasmid pET-32a（＋）,and then transformed into Escherichia coli BL21（DE3） with IPTG induction.SDS-PAGE analysis showed that the fusion protein with molecular weight of 64.2 ku was expressed mainly in insoluble fractions.The IPTG induction time,concentration and temperature were optimized to be 6 h,1.2 mmol/L and 24 to 27 ℃,respectively.Then the inclusion bodies were purified to homogeneity by Ni2＋-affinity chromatography and it could be recognized by the MDRV positive serum in Western-blot assay,which indicated that the purifiedσA proteins still had high reactionogenicity.The purifiedσA protein was then injected into New Zealand rabbit for preparation of polyclonal antibody,the ELISA titer of antiserum was approximately up to 1 ∶ 32 000.Western-blot analysis further confirmed that the prepared polyclonal antibody could react specifically with MDRV-σA protein,which would lay a foundation for further functional study on MDRV σA protein.

关 键 词：番鸭呼肠孤病毒 σA蛋白 原核表达 多克隆抗体 

分 类 号：S852.659.4[农业科学—基础兽医学]