食管疾病2025，Vol.7Issue(3)：182-187,6.DOI:10.15926/j.cnki.issn2096-7381.2025.03.004
重组T7噬菌体溶菌酶的纯化及其牙龈卟啉单胞菌溶菌活性研究
Purification of Recombinant T7 Bacteriophage Lysozyme and Its Lytic Activity Against Porphyromonas gingivalis
摘要
Abstract
Objective To produce T7 lysozyme(T7-Lys)from Escherichia coli T7 double-stranded DNA bacteriophage through genetic engineering and to evaluate the enzyme's antibacterial activity against Escherichia coli and Porphyromonas gingivalis(Pg).Methods The gene encoding T7 lysozyme(T7-Lys)was synthesized based on the T7-Lys gene sequence from GenBank,with codon optimization according to the preference of Escherichia coli.The synthesized gene was cloned into the prokaryotic expression vector pET-28a(+),and the recombinant plasmid pET-28a(+)/T7-Lys was constructed,which was then transformed into Escherichia coli BL21(DE3)for expression.The IPTG concentration and Escherichia coli culture temperature were optimized,and the expression of the soluble target protein was analyzed using SDS-PAGE to determine the optimal culture and induction conditions.T7-Lys was purified through affinity chromatography,and its lytic activity against Escherichia coli and Porphyromonas gingivalis was evaluated at various concentrations.Results The pET-28a(+)/T7-Lys recombinant expression plasmid was successfully constructed and verified by double enzyme digestion and sequencing analysis.Using the ProtParam tool from Expasy,the molecular weight of the recombinant protein was predicted to be approximately 19.15 kDa,with a grand average of hydropathicity(GRAVY)of-0.565.The optimal IPTG induction conditions were determined to be a concentration of 0.5 mmol·L-1 at 23 ℃,with an induction time of 24 hours.The recombinant T7-Lys protein was predominantly expressed in a soluble form.Laboratory-scale fermentation yielded approximately 79.26 mg of purified T7-Lys protein per liter of fermentation broth.T7-Lys has a significant lytic activity against Escherichia coli with a specific activity of 29 081 U·mg-1,and a more pronounced lytic activity against Porphyromonas gingivalis with a specific activity of 35 948 U·mg-1.Conclusion The recombinant plasmid pET-28a(+)/T7-Lys was successfully constructed,leading to the production of soluble T7-Lys protein.T7-Lys exhibited significant lytic activity against Gram-negative bacteria,including Escherichia coli and Porphyromonas gingivalis.关键词
牙龈卟啉单胞菌/大肠杆菌/重组T7溶菌酶/诱导表达/亲和层析Key words
Porphyromonas gingivalis/Escherichia coli/recombinant T7 lysozyme/induced expression/affinity chromatography分类
医药卫生引用本文复制引用
李研研,田重庆,郝雨杭,左嘉慧,张一鸣,陶星宇,李智涛,高社干..重组T7噬菌体溶菌酶的纯化及其牙龈卟啉单胞菌溶菌活性研究[J].食管疾病,2025,7(3):182-187,6.基金项目
河南科技大学大学生科研训练计划(SRTP)资助项目(2023362) （SRTP）
