摘要
目的:对一株新型普通变形杆菌噬菌体中的裂解酶Lys66进行基因克隆、蛋白表达纯化及活性分析.方法:将噬菌体全基因序列在Genbank数据库中进行对比,挖掘出裂解酶基因序列,并对其进行克隆,进一步在大肠杆菌中表达蛋白并纯化,探究其抑菌效果.结果:通过比对挖掘出与裂解酶相似度较高的基因序列,大小为393 bp;利用ExPAsy Bioinformatics Resource Portal预测裂解酶的分子质量为 15.20 kDa,等电点为 9.40,由 130个氨基酸组成,将优化的合成基因构建到载体pET-32α中,得到重组质粒pET-32α-lys66,并将重组质粒转入大肠杆菌BL21(DE3)感受态细胞后诱导其表达,经纯化验证后,获得 1.86 mg/mL的重组裂解酶Lys66蛋白.裂解酶Lys66在平板上的抑菌圈直径为 19.30 mm;对 15株受试菌株中的 13株经氯仿处理的革兰氏阴性菌均表现出裂解活性,宿主谱较广.Lys66(1.89 mg/mL)与乙二胺四乙酸(1 mmol/L)联用,2 h后OD600 nm下降 0.61,抑菌效果较好.结论:本研究重组表达的裂解酶Lys66具有良好的抑菌效果,可作为一种潜在的抗菌剂.
Abstract
Objective:The gene cloning,protein expression,purification and activity analysis of a new type of Proteus vulgaris bacteriophage lyase Lys66 were performed.Methods:The whole gene sequence of bacteriophage was compared in the Genbank database.The gene sequence of lysase was excavated and cloned.The protein was expressed in Escherichia coli and was further purified to explore its antibacterial effect.Results:A gene sequence with high similarity to lyase was discovered through comparison,with a size of 393 bp.By using ExPAsy Bioinformatics Resource Portal,the lyase was predicted that its molecular weight was 15.20 kDa,the isoelectric point was 9.40,and it was composed of 130 amino acids.The whole optimized synthetic gene was constructed onto vector pET-32α to obtain the recombinant plasmid pET-32α-Lys66.The recombinant plasmid was transferred into competent cells of E.coli BL21(DE3)to induce its expression.After purification and validation,1.86 mg/mL Lys66 protein was obtained.The diameter of the bacteriostatic ring of Lys66 lyase on the plate was 19.30 mm.Thirteen Gram-negative bacteria out of 15 tested strains treated with chloroform showed lytic activity,with a wide host spectrum.When Lys66(1.89 mg/mL)was used in combination with ethylene diamine tetraacetic acid(1 mmol/L),the OD600 nm decreased by 0.61 after 2 h,indicating a good antibacterial effect.Conclusion:The recombinant lysase Lys66 expressed in this study had good antibacterial effects and could be used as a potential antibacterial agent.
基金项目
"十三五"国家重点研发计划重点专项(2018YFD0500500)
西南民族大学中央高校基本科研业务费专项(2020NTD04)
维普
万方数据