【摘要】 根据Z26520中fedF序列设计1对引物,从发病仔猪的腹泻粪便分离获得的大肠杆菌中扩增得到fedF基因,经纯化、连接、转化,将其成功克隆到pMD18-T中,所克隆fedF基因序列与GenBank中收录的其他fedF基因序列的同源性达97.8%～99.2%.另设计1对引物,利用基因工程方法将所克隆的fedF的编码序列亚克隆到表达载体pBV220中,转化大肠杆菌BL21(DE3)获得表达重组菌.测序结果表明,插入编码序列与预期序列一致.SDS-PAGE电泳及Western-blotting分析表明,重组菌在诱导后可以表达特异性的相对分子质量为30 100的FedF蛋白.更多还原

【Abstract】 According to the fedF gene sequence of F18 accessed on GenBank,a pair of primers was designed.Then fedF gene products were amplified from field strain isolated from piglet suffered from diarrhea.After purification,ligation and transformation,fedF gene was successfully cloned into pMD18-T,the cloned gene was confirmed to have 97.8%～ 99.2% identity in gene sequence level with other fedF gene in GenBank.Then the cloned fedF gene was subsequently sub-cloned into expressing vector pBV220 by using another set of primers via biological engineering methods and the resulting recombinant plasmid was then transformed into BL21(DE3) bacteria.It was confirmed by SDS-PAGE analysis and Western-blotting that the recombinant BL21 bacteria induced by IPTG were able to express the FedF protein with molecule weight of 30 100.更多还原