摘要
[目的]以鸭疫里默氏菌(Riemerella anatipestifer)噬菌体CRP2为对象,研究尾丝蛋白(Tail fiber protein,TFP)对噬菌体吸附功能的影响.[方法]克隆ORF70假定的TFP基因,构建重组表达载体pET28a(+)-TFP,在大肠杆菌E.Coli BL21(DE3)中诱导表达,通过Ni-NTA亲和层析纯化.酶联免疫吸附试验(ELISA)测定重组蛋白与CRP2噬菌体的宿主菌外膜蛋白(Outer membrane protein,OMP)的结合作用.竞争吸附试验测定重组蛋白对CRP2噬菌体吸附率的影响.[结果]构建了pET28a(+)-TFP重组表达质粒,在大肠杆菌中得到了可溶性表达.纯化的重组蛋白可以与宿主菌的OMP结合,也可以抑制CRP2对其宿主菌的吸附.[结论]ORF70编码的TFP是噬菌体CRP2的一种受体结合蛋白,其对应的受体是宿主菌的OMP.
Abstract
[Objective]Role of tail fiber protein(TFP)plays in adhering Riemerella anatipestifer by CRP2 phage was investigated.[Method]The putative TFP gene of ORF70 was cloned.The recombinant expression vector pET28a(+)-TFP was constructed and induced to express in E.coli BL21(DE3).The recombinant protein was purified by Ni-NTA affinity chromatography.ELISA was used to determine the binding effect of the recombinant protein with the outer membrane protein(OMP)of host bacteria.A competitive adsorption test was performed to determine the adhering effect of the CRP2 protein.[Result]The recombinant plasmids of pET28a(+)-TFP was successfully constructed and expressed in E.coli.The purified recombinant protein rTFP could bind to the OMP of or suppress the CRP2 adsorption to the host bacteria.[Conclusion]The TFP encoded by ORF70,one of the receptor binding proteins of CRP2 phage,was confirmed to adhere to the host bacteria OMP facilitating the subsequent bactericidal effect.
基金项目
福建省科技计划公益类专项(2021R10260011)
福建省农业高质量发展超越"5511"协同创新工程项目(XTCXGC2021012)
福建省农科院科技创新团队建设项目(CXTD2021005-1)
维普
万方数据