人源抗菌肽LL-37在毕赤酵母中的高效表达及其活性检测

High Expression of the Human Antibacterial Peptide LL-37 in Pichia pastoris and the Detection of Its Activity

摘要:

根据GenBank CAA86115中的LL-37氨基酸序列, 选择毕赤酵母偏好密码子, 采用SOE方法合成了人源抗菌肽LL-37基因。所合成的LL-37基因全长为141 bp, 并在其N端引入kex2裂解位点, 以保证表达抗菌肽具有天然N端。基因克隆入pPICZa-A质粒, 构建分泌型重组酵母表达载体pPICZa-A-LL-37。pPICZa-A-LL-37经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。PCR鉴定为阳性的酵母转化子经甲醇诱导分泌LL-37于发酵上清液, 其表达量为206 mg/L。表达产物LL-37耐热性强, 在100℃条件下40 min内抗菌活性不变, 煮沸3 h以上仍具有活性。琼脂糖孔穴扩散法检测显示LL-37对多种革兰氏阴性菌和阳性菌均具有很好的抑制活性, 其对金黄色葡萄球菌 CowanⅠ(Staphylococcus aureus)、致病性大肠杆菌K99(Enteropathogenic E.coli)和鸡白痢沙门氏菌(Salmonella pullorum)的最小抑菌浓度(Minimal Inhibitory Concentration, MIC)分别为1.56 mg/mL、3.12 mg/mL和1.56 mg/mL。

Abstract:

Based on the gene sequences encoding human antibacterial peptide LL-37 as registered in Gen- Bank (serial number in GenBank is CAA86115), using the preferential condon of P. pastoris, the antibacterial peptide LL-37 gene 141 bp in length was designed and synthesized. Especially a Kex2 signal cleavage site was fused in 5′end of the antibacterial peptide gene. The modified antibacterial peptide gene was clonedinto the pPICZa-A vector to construct the recombinant expression vector pPICZa-A-LL-37. The SacⅠlinearized plasmid pPICZa-A-LL-37 was transformed into P. pastoris X-33 by electroporation. The transformants were identified by PCR using R2 and 5’ AOX1 specific primers. The concentration of the secreted LL-37 was 206 mg/L. The new peptide, which has a weight of 4.5 kD, could remain its inhibition activity after being treated for more than 3 hours in boiled water. Agrose diffusion assay showed that LL-37 had broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria, the MIC to Staphylococcus aureus (CowanⅠ), E. coli K99 and Salmonella pullorum were 1.56 mg/mL, 3.12 mg/mL and 1.56 mg/mL, respectively.