Cloning and expression of Cathelicidins Bac5 gene of dairy cattle and antimicrobial activity of recombinant protein | 奶牛Cathelicidins Bac5基因的克隆、表达及抑菌活性分析
2012
Yang Liheng, Gansu Agricultural University, Lanzhou (China), College of Veterinary Medicine | Zhang Yong, Gansu Agricultural University, Lanzhou (China), College of Veterinary Medicine | Xu Peng, Gansu Agricultural University, Lanzhou (China), College of Veterinary Medicine
Chinese. 根据已发表的牛Bac5 mRNA序列设计1对特异性引物，进行RT-PCR扩增，将目的片段定向克隆到原核表达载体PET28a（＋），构建原核重组表达质粒PET28a-Bac5，并转化大肠杆菌Rosetta，进行IPTG诱导和SDS-PAGE检测。结果表明：采用RT-PCR技术成功扩增出414bp去信号肽的Bac5基因片段，重组蛋白经ZPTG诱导和SDS-PAGE检测发现，在19ku附近有1条清晰蛋白条带，与理论预测值大小一致；对表达的Bac5重组蛋白（rBac5）处理后进行抑菌活性的初步鉴定结果表明，rBac5对大肠杆菌（CVCC1450）和金黄色葡萄球菌（CVCC545）2种标准菌株和乳房炎乳的3种常见分离菌株均有一定的抑菌活性。
Show more [+] Less [-]English. A pair of primers were designed and synthesized based on the published Bac5 mRNA sequence of dairy cattle, then the 414 bp Bac5 gene without signal sequence was amplified by RT-PCR. The target gene was cloned into PET28a（＋）, and prokaryotic expression plasmid PET28a-Bac5 was constructed and transformed into E.coli Rosetta, Bac5 was expressed under the induction of IPTG. There was a clear protein band near the 19 ku identified by SDS-PAGE. The antimicrobial activity of expressed recombinant protein（rBac5） was identified by antimicrobial test, the result suggested that the recombinant protein had some antimicrobial activity to two standard strains and the three main strains separated from mastitis milk.
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