抗菌肽LL-37在毕赤酵母SMD1168中的高效表达及活性鉴定

High expression of antimicrobial peptide LL-37 in yeast (SMD1168)

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AvrII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237mg/L。表达产物耐热性强,在100℃条件下30min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1.56μg/mL、3.12μg/mL和6.25μg/mL。

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 diffusion assay showed that LL-37 had potent antibacterial activity against both Gram-negative and Gram-positive bacteria, with MIC of 1.56 μg/mL, 3.12 μg/mL and 6.25 μg/mL against E. coli DH5α, E. coli D31 and Streptococcus suis, respectively.