杂合抗菌肽Me-HNP1真核表达载体的构建与初步表达

Construction and Preliminary Expression of Eukaryotic Expression Vector of Hybrid Antibacterial Peptide Me-HNP1

选择蜂毒肽(Melittin)和人体防御素(HNP-1)部分基因序列进行人工融合,通过生物信息学技术预测其理化性质和功能结构,依据毕赤酵母(Pichia pastoris)密码子偏爱性原则编码杂合肽Me-HNP1基因,聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增目的基因并定向克隆至穿梭型表达载体pPICZa A上,将构建成功的分泌表达载体pPICZαA-Me-HNP1通过电转化的方式转化到毕赤酵母感受态细胞GS115内,以甲醇为诱导剂进行诱导表达,将得到的粗蛋白进行分离纯化。Tricine-SDS-PAGE蛋白电泳在5 kDa处得到一条单一清晰条带,表明杂合抗菌肽Me-HNP1在毕赤酵母中成功表达,生物信息学分析得出杂合抗菌肽Me-HNP1分子内带有8个正电荷,等电点为9.55,脂溶性80.44,在酵母体内半衰期大于20 h。表明其具有成为优秀抗菌肽潜力。为抗菌肽的研究奠定了实验基础。

Select partial gene sequences of Melittin and Human Defensin(HNP-1)for artificial fusion,and their physical and chemical properties,functional structure were predicted by bioinformatics.According to the Pichia pastoris codon preference principle,encoded the hybrid peptide Me-HNP1 gene,and polymerase chain reaction(PCR)amplified of the target gene and directional cloning to the shuttle type expression vector on pPICZa A.The constructed secreted expression vector pPICZαA-Me-HNP1 was transformed into Pichia pastoris competent cell GS115 by electroporation and induced to express with methanol as the inducer.The obtained crude protein was isolated and purified.Tricine-SDS-PAGE protein electrophoresis obtained a single clear band at 5 kDa.The antimicrobial peptide Me-HNP1 was successfully expressed in Pichia pastoris.Bioinformatics analysis revealed that the hybrid antibacterial peptide Me-HNP1 had 8 positive charges in its molecule,theoretical pI:9.55,aliphatic index:80.44 and a half-life of more than 20 hours in yeast.It showed that it had the potential to become an excellent antibacterial peptide,which laid the experimental foundation for the subsequent study of antimicrobial peptides.