一种新融合抗菌肽Hex-Mag基因的克隆、表达及其抗菌活性的研究

Cloning,expression and characterization of a new hybrid AMP gene of Hex-Mag

摘要:

通过在Magainin1-12(GIGKFLHSAKKF)的N端添加碱性氨基酸片段Hexapeptide(RRWQWR)以增强其对细胞膜的吸附能力来提高Magainin1-12的抗菌活性。利用Hexapeptide和Magainin1-12的基因序列,结合酵母偏爱密码子设计出新的融合基因Hex-Mag,通过重叠区扩增基因拼接法(Gene splicing by overlap extension,gene SOEing)利用PCR扩增出基因片段,再将融合基因进行酶切并纯化后导入穿梭质粒pPIC9中,构建受乙醇氧化酶1基因(AOX1)的启动子与转录终止区控制的酵母表达质粒,转化GS115毕赤酵母宿主菌,经表型筛选,阳性克隆用甲醇诱导表达。表达出的融合肽Hex-Mag分子量约2.3kDa,其耐热性强,在100℃条件下,其活性可维持3h以上。琼脂糖孔穴扩散法检测显示Hex-Mag对多种革兰氏阴性菌和阳性菌具有抑制活性,与Magainin1-12相比,其活性有明显增强,N端正电荷增加的预期效应得到初步体现。

Abstract:

To enhance the antibacterial ability of Magainin1-12，its N side was joined with an alkaline peptide named Hexapeptide(RRWQWR)，which would make Magainin1-12 cling to the membrane of bacterial cells even tighter. According to the partiality codon of Pichia pastoris，a new hybrid antibacterial peptide Hex-Mag was designed based on the sequence of Hexapeptide and Magainin(1-12). Synthesized through gene splicing by overlap extension,the hybrid gene was cloned into pPIC9 to construct the expression vector pPIC9-HM. After restriction enzyme analysis and purification, the pPIC9-HM was transformed into Pichia pastoris GS115. And the positive clones screened by the phenotype were induced by methanol. After optimized the requirements for the flask-shaking culture fermentation，the hybrid antibacterial peptide was expressed on high level. The new peptide，which has a weight of 2.3kDa，could remain its inhibition activity after treating for more than 3 hours in boiled water. Detected by agrose diffusion assay，Hex-Mag showed its broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria. The function of additive positive charges were testified by the antibacterial experiments, and the results showed the activity of Hex-Mag was stronger than that of Magainin1-12 obviously.