抗菌肽SMAP-29基因密码子优化及其在毕赤酵母中的表达

目的]对抗菌肽SMAP-29的基因密码子进行优化,并使其在毕赤巴斯德酵母(Pichia pastoris)中表达。方法]根据已知抗菌肽SMAP-29的氨基酸序列,参照毕赤巴斯德酵母密码子的偏好性,设计合成抗菌肽SMAP-29成熟肽基因片段,并同载体pPIC3.5K连接,电击转化至毕赤酵母受体菌GS115中,经G418筛选高拷贝转化子,并用MM、MD板和PCR法筛选Mut+表型。用甲醇诱导表达构建好的重组酵母表达基因工程菌,并裂解酵母细胞进行Tricine-SDS-PAGE分析。结果]在诱导至第2天的细胞裂解液中检测到与预测的SMAP-29分子量相当,约为3.2 kD的表达带;表达产物对金黄色葡萄球菌和白色念珠菌有明显的抑菌作用,而对大肠杆菌抑菌效果不明显。结论]该研究为SMAP-29在生物医学、农业等领域中的应用奠定基础。

Codon Optimization of SMAP-29 Gene and Its Expression in Pichia pastoris

Objective] This study aimed to optimize the codon of antimicrobial peptide SMAP-29 gene and express SMAP-29 in Pichia pastoris.Method] According to the published amino acid sequence of SMAP-29,a gene encoding SMAP-29(sheep myeloid antibacterial peptides-29) mature peptide was synthesized and was biased codon usage of Pichia pastoris.The synthesized gene was inserted into Pichia pastoris expression vector pPIC3.5K,transformed into Pichia pastoris strain GS115 by electroporation,and screened with G418 for high-copy recombinants.The methanol utility plus phenotype(Mut+) was selected with MM,MD and PCR.The correctly constructed recombinant strain was induced with methanol.Expression of SMAP-29 was analyzed by lysing the induced yeast cells with Tricine-SDS-PAGE.Result] After induced with methanol for 2 d,a 3.2 kD induced band was detected in the cell lysate which was consistent to the predicted molecular weight of SMAP-29;the expression products purified with gel filtration chromatography showed significant antibacterial effect against Staphyloccocus aureus and Monilia albican but no significant antibacterial effect against Escherichia coli.Conclusion] This study laid the foundation for the application of SMAP-29 in biomedicine,agriculture and other fields.