摘要
采用SOE-PCR技术,设计合成hLF18-40的编码基因;以融合表达策略构建了pET-43.1a-hLF18-40表达质粒,并获得较高的可溶融合蛋白表达;将融合蛋白Nus-hLF18-40进行Ni2+柱亲和层析、除盐、浓缩后,用肠激酶切割释放抗菌肽hLF18-40,为今后基因工程法表达人乳铁蛋白肽提供了依据。同时,将hLF18-40的C末端氨基酸逐一缩短直至hLF18-32,用化学法合成hLF18-40、hLF18-39、hLF18-38、hLF18-37、hLF18-36、hLF18-35、hLF18-34、hLF18-33和hLF18-32等9条肽,采用肉汤微量稀释法分别测定其最小抑菌浓度(MIC),结果发现:hLF18-40的MIC为40μmol.L-1,hLF18-39的MIC略微下降(20～40μmol.L-1),hLF18-35的MIC与hLF18-40的相同。
In this study,after designing gene sequence,the gene fragment of hLF18-40 was obtained through gene SOE-PCR;The DNA sequence was inserted into pET-43.1a（＋）vector;The constructed vector pET-43.1a-hLF18-40 successfully expressed soluble fusion protein,then Nus-hLF18-40 fusion protein was purified by Ni2＋chromatography purification,desalination and concentration,the hLF18-40 fusion protein was cleaved by recombinant enterokinase.This work laid the foundation for the design and filtration for the future researches.In addition,hLF18-40 was culted down one amino acid each time from the C-terminal and ending at hLF18-32.Nine peptides（hLF18-40,hLF18-39,hLF18-38,hLF18-37,hLF18-36,hLF18-35,hLF18-34,hLF18-33 and hLF18-32）were synthesized by chemical method and their antibacterial activities were determined.The results showed that the MIC of hLF18-40 was 40 μmol·L-1,and the antibacterial activity of hLF18-39 was decreased slightly with MIC of 20~40 μmol·L-1,the antibacterial activity of hLF18-35 was the same as that of hLF18-40.
出处
《化学与生物工程》
CAS
2011年第10期28-31,共4页
Chemistry & Bioengineering
基金
国家863计划资助项目(2007AA100602)
关键词
人乳铁蛋白肽
融合表达
纯化
C端缩短
抗菌活性
human lactoferricin
fusion expression
purification
shorten C-terminal
antibacterial activity
