拟穴青蟹抗菌肽SCY2在毕赤酵母中的表达及其抗菌活性

为深入研究拟穴青蟹Scylla paramamosain阴离子抗菌肽SCY2的抗菌活性,将抗菌肽SCY2的成熟肽序列与毕赤酵母Pichia pastoris的p PIC9K质粒连接,构建了分泌型表达载体p PIC9K-SCY2,用电击法将其转化至毕赤酵母GS115菌株中,经甲醇诱导表达和亲和纯化后获得目的蛋白,并进行质谱鉴定和抗菌活性分析。结果表明:本研究中成功构建了重组毕赤酵母菌株GS115/p PIC9K-SCY2,用0.5%甲醇诱导表达24 h后,分泌表达上清液中获得了相对分子质量约11 000的稳定表达产物,与预期的抗菌肽SCY2成熟肽的大小相符;纯化后的目的蛋白经质谱鉴定,有6个肽段的氨基酸序列与预期一致;抗菌活性结果表明,抗菌肽SCY2能抑制革兰氏阳性菌生长,对被测的革兰氏阴性菌无抑杀活性(50μmol/L);抗菌肽SCY2同抗生素进行协同抗菌试验,发现SCY2与四环素协同使用对荧光假单胞菌Pseudomonas fluorescens的抑杀存在协同作用,SCY2与青霉素协同使用对施氏假单胞菌Pseudomonas stutzeri的抑杀具有增强作用。研究表明,较低浓度的抗生素与抗菌肽协同使用可有效地减少抗生素的使用,降低食品中抗生素的残留风险。

Expression,purification and antimicrobial activity of SCY2 from mud crab Scylla paramamosain in yeast Pichia pastoris

A 300 bp cDNA sequence of SCY2,as an anionic antimicrobial peptide recently identified from the se-minal plasma of mud crab Scylla paramamosain,encoding mature peptide,was cloned into yeast Pichia pastoris by the secreted expression vector of pPIC9K, and then induced by methanol to gain more detailed information on its antimicrobial activity. The recombinant product was identified using MS-fingerprinting. The results showed that re-combinant plasmid pPIC9K-SCY2 was constructed successfully, and the clone of GS115/pPIC9K-SCY2 induced by 0.5% methanol for 24 h,with a stable expressed product with molecular weight of about 11 000,whose molecu-lar weight was consistent with the calculated molecular weight of SCY2.The purified recombinant protein was identi-fied by mass spectrum analysis,and 6 peptide segments of recombined SCY2 were sequenced correctly using mass spectrum. The recombinant product of SCY2 was shown to have the activity against Gram-positive bacteria,without activity against Gram-negative ones(>50 μmol/L). Synergistic antibacterial test of SCY2 with penicillin,strepto-mycin and tetracycline revealed that SCY2 combined with tetracycline had synergistic antibacterial function against Pseudomonas fluorescens,especially combination of SCY2 with penicillin against Pseudomonas stutzeri. The findings indicate that the combined use of low concentrations of antibiotics and antimicrobial peptides can effectively reduce the amount of antibiotics and the risk of antibiotic residues in food.