噬菌体裂解酶Cp51在重组乳酸菌中的表达及对A型产气荚膜梭菌的抗菌活性

Expression of phage lysin Cp51 in recombinant Lactococcus lactis and its antibacterial activity against Clostridium perfringens type A

【目的】构建A型产气荚膜梭菌Clostridium perfringens裂解酶重组表达乳酸菌Lactococcus lactis,并检测重组菌及其表达产物的抗菌效果。【方法】全基因合成噬菌体裂解酶Cp51基因,构建PNZ8148-Cp51原核表达重组载体,电转化至乳酸菌NZ9000,经乳链菌肽诱导表达可溶性Cp51蛋白;用比浊法检测表达蛋白对A型产气荚膜梭菌的抗菌活性;利用细菌液混合培养检测重组菌对A型产气荚膜梭菌增殖的抑制作用。【结果】噬菌体裂解酶Cp51在乳酸菌中成功表达,为1268 bp;质量浓度1μg·mL^−1以上的重组蛋白对A型产气荚膜梭菌具有高效抗菌活性;重组乳酸菌对A型产气荚膜梭菌增殖有一定的抑制效果,混合培养后使产气荚膜梭菌活菌数从9×10^9 CFU·mL^−1下降到约9×10^8 CFU·mL^−1。【结论】A型产气荚膜梭菌噬菌体裂解酶重组乳酸菌构建成功,为后续优化表达和临床应用研究奠定了基础。

【Objective】To construct a recombinant Lactococcus lactis expression system of phage lysine Cp51,and study its antibacterial activity against Clostridium perfringensin type A in vitro.【Method】Phage lysin Cp51 gene was inserted into the prokaryotic expression vector PNZ8148,and then the recombinant vector PNZ8148-Cp51 was constructed and transformed into L.lactis NZ9000.Nisin was used to induce expression of soluble protein Cp51.The antibacterial activity of the recombinant protein against C.perfringens type A was detected by kinetic turbidimetric assay in vitro.The inhibitory effect of recombinant L.lactis on the proliferation of C.perfringens type A was tested by mixed cultivation.【Result】The recombinant phage lysin Cp51 was successfully expressed in L.lactis and the Cp51 gene was 1268 bp in length.The recombinant protein had strong antibacterial activity against C.perfringens type A at the concentration above 1μg·mL^−1 in vitro.The recombinant L.lactis also had certain inhibitory effect on C.perfringens type A with the number of C.perfringens decreased from 9×10^9 CFU·mL^−1 to 9×10^8 CFU·mL^−1 after mixed cultivation.【Conclusion】Recombinant L.lactis expressing phage lysin Cp51 which against C.perfringens type A has been successfully constructed,which provides a basis for further optimization of expression and clinical application.