人ⅡA型磷脂酶A2衍生多肽M17杀菌作用的研究

Study on Bactericidal Activity of the Polypeptide M17 Derived from Human Group ⅡA Phospholipase A2

摘要:

目的: 研究和比较人ⅡA型磷脂酶A₂(phospholipase A₂,PLA₂)衍生多肽M17,P26和N_1-11对不同细菌在体外的杀菌效应。方法: 根据人ⅡA型PLA₂氨基酸顺序分别设计合成3个肽分子M17(p_51-67),P26(p_99-124),N_1-11(p_1-11)。采用琼脂铺板计数法测定杀菌活性,将不同浓度衍生肽分别与革兰阳性菌(G⁺)金黄色葡萄球菌、枯草杆菌、屎肠球菌和革兰阴性菌(G^-)大肠杆菌、绿脓杆菌、鲍曼不动杆菌在37℃孵育2.5 h,然后铺板并置于37℃恒温箱培养18~24 h,记录每一琼脂板上的菌落数(CFU),并计算多肽作用后的杀菌率。结果: ⅡA型PLA₂M17对枯草杆菌、金黄色葡萄球菌、屎肠球菌的杀菌回归方程分别为:Y=1.464X+3.795(ED₅₀ 6.65 mg·L^-1),Y=1.152X+3.419(ED₅₀ 23.57 mg·L^-1),Y 0.836X+3.832 (ED₅₀ 24.95 mg·L^-1);对大肠杆菌、鲍曼不动杆菌、绿脓杆菌的杀菌回归方程分别为:Y=0.877X+4.054(ED₅₀ 12.00 mg·L^-1),Y=1.094X+3.371(ED₅₀ 30.83 mg·L^-1),Y=1.027X+3.626(ED₅₀ 21.77 mg·L^-1);M17肽分子对G⁺菌与G^-菌均有较强的杀菌活性。P26和N_1-11对金黄色葡萄球菌的杀菌回归曲方程分别为:Y=0.915X+2.766(ED₅₀ 276.39 mg·L^-1),Y=1.389X+1.668(ED₅₀ 250.52 mg·L^-1), P26和 N_1-11对大肠杆菌的杀菌回归方程为:Y=1.327X+1.437(ED₅₀ 484.18 mg·L^-1), Y=0.881X+2.903(ED₅₀ 240.02 mg·L^-1);N_1-11和P26对G⁺菌有较强的杀菌活性,但对G^-菌杀菌活性较弱。M17肽分子对金黄色葡萄球菌的杀菌活性强于P26及N_1-11,M17肽分子对大肠杆菌的杀菌活性明显强于P26及N_1-11。结论: 衍生自人ⅡA型PLA₂保守区域17个氨基酸残基的肽M17对G⁺菌和G^-菌均有较强的杀菌活性,这可能与M17肽分子更易于与细菌结合并发挥作用有关。

Abstract:

Objective: To study and compare the bactericidal activity of the polypeptide M17, P26 and N_1-11 which derives from hunman group ⅡA phospholipase A₂ (PLA₂, phospholipase A₂) in vitro. Method: Peptide M 17(p_51-67), P26 (p_99-124), N_{1– 11} (p_1-11) were synthesized according to the sequence of human GroupⅡA PLA₂ amino acid. Six species of bacterial, Gram positive(G⁺) Staphy 1ococcus aureus, Bacillus subtilis, Enterococcus faecium, Gram negative(G^-) Escherichia coli , Acinetobacter baumannii, Bacillus pyocyaneus , were incubated with different concentration of polypeptides at 37℃ for 2.5 hours in a water bath respectively. Then the reaction solution was diluted and agar plates were poured. After 18-24 hours incubation in the thermostated container at 37℃.The colony formed units were counted and the bactericida1 rates were calculated. Result: Regression equation of PLA₂M17 bactericidal activity on the G⁺ bacteria(lococcus aureus, Bacillus subtilis, Enterococcus faecium) were Y=1.464X+3.795(ED₅₀ 6.65 mg·L^-1),Y=1.152X+3.419(ED₅₀ 23.57 mg·L^-1), Y=0.836X+3.832 (ED₅₀ 24.95 mg·L^-1). Regression equation of M17 bactericidal activity on the G^-bacteria (E. coli , A. baumannii, B. pyocyaneus)were Y=0.877X+4.054(ED₅₀ 12.00 mg·L^-1),Y=1.094X+3.371(ED₅₀ 30.83 mg·L^-1),Y=1.027X+3.626(ED₅₀ 21.77 mg·L^-1). PLA₂M17 possessed potent bactericidal activity on the G⁺ bacteria and G^-bacteria.Regression equation of N_1-11 and P26 bactericidal activity on B. subtilis were Y=0.915X+2.766(ED₅₀ 276.39 mg·L^-1),Y=1.389X+1.668(ED₅₀ 250.52 mg·L^-1). Regression equation of N_1-11 and P26 bactericidal activity on E. coli were Y=1.327X+1.437(ED₅₀ 484.18 mg·L^-1), Y=0.881X+2.903(ED₅₀ 240.02 mg·L^-1).N_1-11 and P26 possessed strong bactericidal activity of G⁺ bacteria, but weaker activity of G^- bacteria. M17 possessed stronger bactericidal activity than N_1-11 and P26 on B. subtilis and E. coli. Conclusion: Peptide M17 derived from human Ⅱ type A PLA₂ highly conserve region possesses a strong bactericidal activity, this may be related to the M17 peptide molecules more easily binding to bacteria.