摘要
牛乳铁蛋白活性多肽(Lactoferricin B,LfcinB)是一种阳离子型抗菌肽,因其具有多种生物学功能,现已被认为具有能够替代传统抗生素的发展前景。然而,由于传统的分离制备方法存在诸多弊端,所以基于基因工程的原核表达技术在制备高纯度和高效表达的该蛋白方面具有极其深远的意义。研究旨在摸索出一套完整的利用原核表达技术制备LfcinB的分子生物学方法。依据大肠杆菌密码子偏爱性的原则,设计了LfcinB的基因序列,通过人工合成的方法获得该基因的优化序列,借助Bam HI和Sal I双酶切、连接等手段将其构建到原核表达载体pGEX-4T-2上,获得重组表达载体pGEX-4T-2-LCB,将该载体转化E.coli Rosetta(DE3)。使用IPTG诱导,结果发现LfcinB在大肠杆菌中表达量超过20%。通过一系列蛋白纯化技术分离得到纯度达到95%以上的LfcinB融合蛋白。抑菌试验结果表明,重组抗菌肽LfcinB融合蛋白具有很好的抑菌活性。研究结果为人们使用基因工程技术制备抗菌肽LfcinB提供了具有重要参考价值的技术路线和理论依据。
As one kind of cationic antimicrobial peptide,lactoferricin B （LfcinB） with many biofunctions is considered to be a potential alternative to traditional antibiotics.However,the traditional methods showed much inefficiency in the preparation and purification ofLfcinB.Therefore,itis ofthe great significance to carry on the large-scale production of LfcinB through the genetic engineering based on the prokaryotic expression technology.This study aimed to find a set of strategies for the preparation of LfcinB through the prokaryotic expression technology.Forthe purpose of the highly efficient expression of LfcinB gene in E.coli,the original sequence of LfcinB gene was firstly designed based on the codon preference of E.coli and then the optimized sequence was artificially synthesized.The optimized LfcinB gene was digested by Bam HIand SalI,and ligated with pGEX-4T-2 digested the same two restriction enzymes as the above.The new recombinant construct was designated as pGEX-4T-2-LCB.With the induction of IPTG,the LfcinB fused protein was highly efficiently expressed in E.coli Rosetta （DE3） containing pGEX-4T-2-LCB.The amount of LfcinB fused protein was more than 20%of total soluble proteins in E.coli.The LfcinB fused protein with more than 95% purity was finally obtained by affinity chromatography.And the following in vitro antimicrobial test showed that LfcinB fused protein possessed the very good antimicrobial activity.This work provided a valuable technicalroute forthe production ofLfcinB by genetic engineering.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2010年第9期67-71,共5页
Journal of Northeast Agricultural University
基金
国家自然科学基金(30800794)
黑龙江省青年科学基金(QC08C05)
黑龙江省科学院青年基金
关键词
抗菌肽
牛乳铁蛋白活性多肽
融合蛋白
原核表达
antimicrobial peptide
lactoferricin B
fusion protein
prokaryotic expression
