A型产气荚膜梭菌噬菌体裂解酶Cp51的原核表达及活性检测

【目的】原核表达A型产气荚膜梭菌噬菌体裂解酶Cp51并研究其对A型产气荚膜梭菌Clostridium perfringens的体外抗菌活性。【方法】合成噬菌体裂解酶Cp51基因,并构建了pET-32a-Cp51原核表达载体,转化到大肠埃希菌Escherichia coli BL21(DE3),经终浓度为0.5 mmol·L~(-1)的IPTG诱导以及镍柱纯化后,获得了可溶性的Cp51重组蛋白;用比浊法检测Cp51重组蛋白的杀菌活性。【结果】噬菌体裂解酶Cp51重组蛋白能够有效降低7株A型产气荚膜梭菌的浊度,裂解酶质量浓度在5μg·m L~(-1)以上、作用30 min对A型产气荚膜梭菌的杀菌率可达到99.99%以上,而对其他种类细菌无杀菌效果。【结论】A型产气荚膜梭菌噬菌体裂解酶Cp51重组蛋白对A型产气荚膜梭菌有较强的体外杀菌活性和特异性,研究结果为后续裂解酶Cp51的临床应用奠定基础。

Prokaryotic expression and activity detection of bacteriophage lysin Cp51 against Clostridium perfringens type A

Objective] To construct a prokaryotic expression system of type A Clostridiumperfringens phage lysin Cp.51,and study its antibacterial activity against C Perfringens type A in vitro.Method] Bacteriophage lysin Cp51 gene was synthesized.The prokaryotic expression vector pET-32a-Cp51 was constructed and transformed into Escherichia coli BL21 (DE3).After induction using 0.5 mmol·L-1 IPTG,the soluble recombinant protein Cp51 was successfully expressed,and was subsequently purified with Ni2+-NTA affinity chromatography.The antibacterial activity of the recombinant protein Cp51 was detected by kinetic turbidimetric assay.Result] The bacteria turbidities of seven strains of C.perfringens type A were effectively reduced by the recombinant protein Cp51.The bactericidal rate was above 99.99％ in 30 min after treatment of recombinant protein Cp51 at the concentration of above 5 μg·mL-1.The Cp51 protein had no bactericidal effect against other types of bacteria.Conclusion] The recombinant protein of bacteriophage lysine Cp51 has strong bactericidal activity and specificity in vitro against type A C.perfringens,which could provide a basis for clinical application of Cp51 lysin.