噬菌体裂解酶LysP53漱口水的制备与评价

Preparation and Evaluation of Bacteriophage LysP53 Mouthwash

目的:开发一种含有噬菌体裂解酶LysP53的漱口水,并在体外评价其抗菌活性、细胞毒性和稳定性。方法:体外克隆、表达和纯化噬菌体裂解酶LysP53并制备成漱口水。通过杀菌活性测定、结晶紫染色试验、扫描电子显微镜等评价LysP53漱口水的杀菌效果,细胞计数试剂盒-8试验(CCK-8)对其安全性进行体外评估,不同温度条件下对LysP53活性的影响评价其稳定性。结果:8μmol/L LysP53漱口水对主要致龋菌变形链球菌、远缘链球菌、内氏放线菌有杀菌效果(P<0.05),而对常见共生菌口链球菌、轻链球菌、血链球菌抗菌活性较差(P>0.05)。在已形成的生物膜中应用LysP53漱口水,可使生物膜结构崩解,并有效减少活菌数量(P<0.05)。CCK-8显示各组之间细胞活性无统计学差异(P>0.05),LysP53漱口水分别储存于4、37、43、60、95℃1 h后的杀菌效率无差异,且仍显示出优良的杀菌活性。结论:LysP53漱口水表现出良好的抗菌、特异性、稳定性和生物相容性,作为常规口腔卫生措施,在预防龋齿方面具有巨大的潜力。

Objective:To develop a mouthwash containing the bacteriophage lysin LysP53 and evaluate its bactericidal activity,cytotoxicity,and stability in vitro.Methods:LysP53 was cloned,expressed,and purified in vitro and prepared into mouthwash.The bactericidal effect of LysP53 was evaluated by bactericidal activity assay,crystal violet staining test,and scanning electron microscopy.Its safety was assessed in vitro by CCK-8.Stability was evaluated by the effect on LysP53 mouthwash bactericidal activity at different temperatures.Results:8μmol/L LysP53 mouthwash had bactericidal effect on the main cariogenic bacteria Streptococcus mutans,Streptococcus sobrinus,and Actinomyces naeslundii(P<0.05),but had poor antibacterial activity against the common commensal bacteria Streptococcus oralis,Streptococcus mitis,and Streptococcus sanguinis(P>0.05).Application of LysP53 mouthwash in established biofilms disintegrated the biofilm structure and effectively reduced the number of viable bacteria(P<0.05).CCK-8 showed no statistical difference in cell viability between the groups(P>0.05).LysP53 mouthwash showed no difference in bactericidal efficiency after 1 hour of storage at 4,37,43,60,and 95℃,and still showed excellent bactericidal activity.Conclusion:LysP53 mouthwash has good antibacterial activity,safety,and stability.It has great potential in preventing dental caries for routine oral hygiene measures.