抗菌肽Cecropin D在毕赤酵母中的表达、纯化及活性鉴定

目的在毕赤酵母GS115中表达抗菌肽Cecropin D,并检测其抗菌活性。方法根据毕赤酵母密码子的偏爱性,人工合成6条寡聚核苷酸片段,经磷酸化、退火、连接并克隆入酵母表达载体pPIC9K。重组表达质粒pPIC9K-D转化至毕赤酵母菌GS115,经G418筛选,阳性高拷贝克隆用0.5%的甲醇诱导表达,表达产物经CM-Sepharose层析柱纯化,Tricine-SDS-PAGE分析,琼脂糖扩散法和比浊法测定其抗菌活性。结果经Tricine-SDS-PAGE检测,在相对分子质量3900左右处可见目的蛋白表达条带,纯化后的目的蛋白纯度达90%以上,获得的抗菌肽Cecropin D对一些革兰阳性菌和革兰阴性菌均有抑菌活性。结论抗菌肽Cecropin D成功地在毕赤酵母中分泌表达,为以后深入研究抗菌肽奠定了基础。

Expression of Antibacterial Peptide Cecropin D in Pichia pastoris and Purification and Activity of Expressed Product

Objective To express antibacterial peptide cecropin D in Pichin pastoris and determine the activity of expressed product. Methods Six oligonucleotide fragments were synthesized according to the codon bias of Pichia pastoris ,then phosphorylated,annealed,hnked and cloned to expression vector pPIC9K. The constructed recombinant plasmid pPIC9K-D was transformed to Pichia pastoris GS115, and positive clones were screened with G418 for expression under induction of 0.5% methanol. The expressed product was purified by CM Sepharose chromatography, idntified by Tricine-SDS-PAGE, and determined for antibacterial activity by agarose diffusion test and turbidimetry. Results Cecropin D with a relative molecular mass of about 3 900 was expressed and reached a purity of more than 90% after purification. It showed antibacterial activity to both Gram-positive and negative bacteria. Condusion Cecropin D was successfully expressed in Pichia pastoris, which laid a foundation of further study on antibacterial peptide.