抗菌肽LL_37在毕赤酵母SMD1168中的高效表达及活性鉴定.pdfVIP

第32 卷 第2 期 中 国 预 防 兽 医 学 报 Vol. 32 ，No.2 2010 年 2 月 Chinese Journal of Preventive Veterinary Medicine Feb. 2010 抗菌肽LL- 37 在毕赤酵母SMD1168 中的高效表达及活性鉴定 刘德辉，何 俊，林云熊，黄毓茂* (华南农业大学兽医学院，广东广州 510642) 摘 要：为获得高效表达的抗菌肽 LL-37 ，根据人源抗菌肽LL-37 的氨基酸序列, 选择毕赤酵母(Pichia pastoris)密码子的偏嗜性，通过SOEing 法改造合成LL-37 基因片段，克隆到pGAPZαA 质粒中，获得分泌型重组 酵母表达载体pGAPZαA-LL-37 。pGAPZαA-LL-37 通过限制性内切酶Avr II 酶切线性化后，经电穿孔法转入毕赤 酵母细胞SMD1168。经Zeocin 抗性筛选，得到高拷贝转化子，PCR 检测表明LL-37 基因与毕赤酵母染色体稳定 结合。在GAP 启动子调控下，LL-37 蛋白获得分泌表达，其上清表达量约为237 mg/L 。表达产物耐热性强，在 100 ℃条件下30 min 内仍保持一定的抗菌活性。表达产物对大肠杆菌DH5α 、大肠杆菌D31 和猪链球菌的最小 抑菌浓度(MIC)分别为 1.56 μg/mL、3.12 μg/mL 和6.25 μg/mL 。 关键词：抗菌肽LL-37 ；基因设计；高效表达；活性鉴定 中图分类号：S852.5 文献标识码：A 文章编号：1008-0589(2010)02-0098-04 High expression of antimicrobial peptide LL-37 in yeast (SMD1168) LIU De-hui, HE Jun, LIN Yun-xiong ，HUANG Yu-mao* (College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China) Abstract: In order to improve expression of antimicrobial peptide LL-37 in Pichia pastori, LL-37 gene was designed and synthesized to include the partiality codons of P. pastoris based on the gene sequences encoding human antibacterial peptide LL-37. The modified LL-37 gene was cloned into the pGAPZαA vector and transformed into P. pastoris SMD1168 by electroporation. The transformants were screened with zeocin and LL-37 gene stabley intergrated into yeast chromosome was comfirmed. Up to 237 mg/L LL-37 was produced in culture medium. The new peptide could retain its antibacterial activity after being treated for 30 minutes in boiling water. Agrose