新型生物防腐剂——多聚阳离子抗菌肽在毕赤酵母中的表达-遗传.PDF

※生物工程 食品科学 2013, Vol.34, No.05 161 新型生物防腐剂——多聚阳离子抗菌肽 在毕赤酵母中的表达 李 青，周晓宏* (北京理工大学生命学院，北京 100081) 摘  要：在毕赤酵母GS 115 中表达一种新型食品生物防腐剂——多聚阳离子抗菌肽(poly-cationic antib acterial peptide ，PCAP) 。根据毕赤酵母密码子的偏爱性，人工合成编码多聚阳离子抗菌肽与多聚阴离子肽融合肽的DNA 序列，连接并克隆入酵母表达载体pPIC9 ，得到重组表达质粒pPIC9-PCAP ，并转化至毕赤酵母GS115，筛选阳性克 隆子，用体积分数0.5%的甲醇诱导表达，优化表达条件，表达产物用Tricine-SDS分析，在分子质量8kD左右 处可见 目的蛋白表达条带，表明融合肽成功地在毕赤酵母中分泌表达。在pH2.0条件下将表达产物采用胃蛋白酶酶 解，分离得到的碱性多聚阳离子肽对食品腐败微生物枯草芽孢杆菌具有明显的抑菌作用。 关键词：多聚阳离子；抗菌肽；毕赤酵母；防腐剂 Expression of a Novel Poly-Cationic Antibacterial Peptide as a Food Preservative in Genetically Engineered Pichia pastoris * LI Qing，ZHOU Xiao-hong (School of Life Science, Beij ing Institute of Technology, Beij ing 100081, China) Abstract ：A novel polycationic antibacterial peptide (PCAP) as a food preservative was expressed in genetically engineered Pichia pastoris . A fusion peptide gene of a polycationic and polyanionic peptide was synthesized according to the codon of Pichia pastoris, and then cloned into the vector pPIC9 ．The constructed recombinant plasmid pPIC9-PCAP was transformed into Pichia pastoris GS115, and the positive clones were screened on histidine-deficient MD medium and was then expressed under induction by 0.5% methanol in BMMY medium. The expressed fusion peptide was identified as a peptide with molecular weight of 8 kD in Tricine-SDS, and then digested with pepsin at pH2.0 The polycationic peptide was separated and proved to have antibacterial activity against food spoilage microorganism Bacillus subtilis . Key words Pichia pastoris ：polycationic ；antibacteria