抗菌肽Eumenitin序列修饰后的毕赤酵母重组表达载体构建

Construction of the recombinant expression vector of Pichia pastoris with the sequencially modified antimicrobial peptide Eumenitin

为了构建高效低毒的新型抗菌肽,将抗菌肽Eumenitin及其序列修饰后获得4条新型短肽的毕赤酵母真核表达载体,采用生物信息学方法对Eumenitin及其衍生肽的理化参数进行预测,基因序列经毕赤酵母密码子优化后,克隆至pMD19T-simple载体上,鉴定后连接至毕赤酵母表达载体pGAPZαA上,构建的重组表达质粒经Avr Ⅱ线性化后,电转化至毕赤酵母GS115感受态细胞中,涂布于YPDSZ平板上,进行表型筛选和高拷子筛选,并对5条短肽的溶血率进行测定。结果显示:Eumenitin及其衍生肽均为较稳定的疏水性多肽,空间结构以α螺旋为主,且序列中不含有信号肽。经PCR、双酶切鉴定和核苷酸序列测定,成功构建了5条短肽的毕赤酵母重组表达载体,经设计改造后的抗菌肽溶血性均较低。本研究结果为后续进行重组蛋白的表达以及抑菌活性研究奠定了基础,以期获得抗菌活性强、溶血性低、高表达的新型抗菌肽。

The aim of this study was to construct the eukaryotic expression vector of Pichia pastoris of four novel short peptides modified by the basic sequence of an antimicrobial peptide called Eumenitin and the hemolytic activity of the vector was tested.A bioinformatics method was used to forecast the physical and chemical parameters of Eumenitin.The sequence of the gene of the peptide was optimized by Pichia pastoris codon and the gene was cloned into the pMD19T-simple vector and received identification.Then,the recombinant expression plasmid was linked to the Pichia pastoris expression vector pGAPZαA.The recombinant expression plasmid was linearized by AvrⅡ,and was electrotransformed into the Pichia pastoris GS115 competent cells and coated on YPDSZ plates.Finally,phenotypic screening and high copy screening were carried out,and the hemolysis rates of five short peptides were determined.The results showed that all the five short peptides were of hydrophobic polypeptides.Their spatial structure was mainly alpha helix.There was no signal sequence in the peptides.The recombinant expression vectors of five short peptides were successfully constructed by PCR,double digestion and nucleotide sequencing.The hemolysis of the five short peptides was low.This study laid a foundation for further study on the expression of recombinant protein and its antimicrobial activity,in to the hope of obtaining novel antimicrobial peptides with strong activity,low hemolysis and high expression.