重组人肽抗生素hPAB-β及其突变体的活性测定与筛选

Antimicrobial activity detection of recombinant peptide antibiotics hPAB-β and its artificial variants

目的 测定重组人肽抗生素 h PAB- β及其突变体的抗菌活性 ,筛选理想的活性分子。方法 应用平板法和 MIC测定法检测重组表达的 h PAB- β及突变体 h PAB- β38、h PAB- β34对 8株实验室保存菌株及 10株临床分离菌株的杀菌能力 ,并以化学合成的肽抗生素 h PAB- β作对照 ;根据各目的分子抗菌能力的强弱 ,筛选理想的目标分子 ,并分析突变体结构的变化对其功能的影响。结果 重组 h PAB- β及突变体 h PAB- β38与化学合成的天然分子抗菌活性相当 ,对革兰氏阳性菌的 MIC在 12 5～ 2 5 0 μg/ml之间 ,对革兰氏阴性菌的MIC在 31～ 12 5 μg/ml范围内 ;突变体 h PAB- β34活性下降 4倍左右 ;结构分析表明 h PAB- β38与 h PAB- β参与二级结构中 α螺旋和 β片层构成的氨基酸残基完全相同 ,而 h PAB- β34则有显著不同 ,α螺旋少了 2个残基 ,3个 β片层中除 β2与 h PAB- β相同外 ,β1和 β3的构成均多 2个残基 ,推测 h PAB- β34二级结构的改变是其活性降低的主要原因。结论 重组肽抗生素 h PAB- β具有抗菌活性 ,删除 3个端残基的突变体 h PAB- β38活性不变 ,可作为 h PAB- β走向临床应用的一个新侯选者。

Objective To investigate the antimicrobial activities of recombinant peptide antibiotics hPAB-β and its artificial variants. Methods The antimicrobial activities of recombinant peptide antibiotics hPAB-β and its artificial variants hPAB-β38,hPAB-β34 against 8 Lab-kept bacterial strains and 10 clinical isolates were detected by agar diffusion and minimal inhibition concentration (MIC) assay. The structure and function relationship was analyzed based on the interesting molecule′s antimicrobial activities. Results Results showed that the recombinant hPAB-β and hPAB-β38 had the similar antibacterial activities to the synthetic ones,their MICs to Gram negative bacteria were 31～125μg/ml;to Gram positive bacteria were 125～250 μg/ml . The biological activities of the variant hPAB-β34 was 4 times lower than hPAB-β. Molecular modeling shows that the α-helix of hPAB-β34 is shorter by 2 residues than hPAB-β,while β1 and β3 strands are 2 residues longer. The secondary structure change of hPAB-β34 after excessive terminal residues deletion may be the main reason related to its biological activities. Conclusion Both recombinant hPAB-β and hPAB-β38 (deleted 3 N-terminal amino acids from hPAB-β) are of similar antibacterial activities. The results showed that the hPAB-β is a new promising candidate used clinically.