鲍曼不动杆菌噬菌体ZZ1受体结合蛋白gp12的克隆表达及ZZ1 RBP受体性质鉴定  

Cloning,expression of Acinetobacter baumanni phage ZZ1 receptor binding protein gpl2 and identification of ZZ1RB Preceptors

作　　者：王小亭 张向前[2] 张改 王书伟[2] 李振江[2] 陈松建[2] 刘肖[2] 李亚辉 王山梅[3] 靳静[2] WANG Xiaoting;ZHANG Xiangqian;ZHANG Gai;WANG Shuwei;LI Zhenjiang;CHEN Songjian;LIUXiao;LI Yahui;WANG Shanmei;JIN Jing(Huizhou Second Maternal and Child Health Hospital Neonatal Screening Center,Huizhou 516001,Guangdong,China;Department of Pathogen Biology andImmunology,Henan Medical College;Laboratory Department of Henan Provincial People's Hospital)

机构地区：[1]惠州市第二妇幼保健院新生儿疾病筛查中心,广东惠州516001 [2]河南省医学高等专科学校病原生物学与免疫学教研室 [3]河南省人民医院检验科

出　　处：《中国病原生物学杂志》2023年第9期1053-1057,1064,共6页Journal of Pathogen Biology

基　　金：河南省医学科技攻关计划联合共建项目(No.LHGJ20210169);河南省科技厅科技发展计划项目(No.222102310449,No.202102310098);河南省高等学校重点科研项目(No.23B310012)。

摘　　要：目的 为进一步了解噬菌体与宿主菌之间的相互作用机制,对鲍曼不动杆菌噬菌体ZZ1短尾丝蛋白gp12进行克隆表达并对ZZ1 RBP吸附受体进行鉴定。方法 原核表达噬菌体ZZ1受体结合蛋白gp12,根据分子质量、抗原性以及对ZZ1吸附竞争干扰的功能等对表达的重组受体结合蛋白gp12进行鉴定。分别用细菌膜蛋白提取试剂盒和细菌脂多糖提取试剂盒提取鲍曼不动杆菌表面OMP和LPS,采用ELISA的方法对噬菌体ZZ1作用受体性质进行鉴定。分别用蛋白酶K和高碘酸盐对AB09V菌进行处理以破坏细菌表面的蛋白质和LPS,观察噬菌体ZZ1对两种方法处理AB09V菌的吸附效率。结果 PCR扩增ZZ1gp12基因后,成功诱导表达了含6×His标签的ZZ1重组受体结合蛋白gp12,SDS-PAGE显示所表达蛋白的相对分子质量约为55 ku;使用0.5 mmol/L IPTG诱导4h,蛋白表达基本趋于稳定;Western blot显示纯化蛋白可分别被his标签抗体和小鼠抗ZZ1多克隆抗体识别;竞争吸附试验显示,在ZZ1重组gp12蛋白存在的情况下,ZZ1对鲍曼不动杆菌AB09V的吸附效率与对照组的63.5%相比下降42.4%;当ZZ1重组gp37蛋白和ZZ1重组gp12蛋白均存在的情况下,ZZ1对鲍曼不动杆菌AB09V的吸附率下降55.8%。ELISA和进一步验证试验显示ZZ1重组受体结合蛋白均吸附宿主菌AB09V菌表面的LPS。结论 成功表达了噬菌体ZZ1重组受体结合蛋白gp12,ZZ1重组gp37蛋白和ZZ1重组gp12蛋白均吸附宿主菌AB09V表面的LPS。Objective In order to further understand the interaction mechanism between bacteriophages and host bacteria,the clonal expression of the Acinetobacter baumannii bacteriophage ZZ short tail fiber protein gpl2 was performed and the ZZ1 RBP adsorption receptor was identified.Methods Prokaryotic expression of bacteriophage ZZ1 recombinant receptorbinding protein gp12,and it was identified frommolecular mass,antigenicity and function of ZZl adsorption competition interference.OMP and LPS on the surface of A.baumannii were extracted respectively by Bacterial Membrane Protein Extraction Kit and Bacterial Lipopolysaccharide Extraction Kit,and then identified hostreceptorsforbacteriophage ZZ1 adsorption using ELISA.We handled ABo9V bacteria with proteinase K and periodate to destroy the protein and LPS on the bacterial surface,and observed the adsorption efficiency of the phage ZZ1 on the ABo9V bacteria treated by the two methods.Results After PCR amplified the ZZ1gp12 gene,the ZZ1 recombinant receptorbinding protein gpl2 containing the 6 His tag was successfully induced.SDS-PAGE showed that the relative molecular mass of the expressed protein was about 55 ku.The expression of protein induced at 37 C with 0.5 mmol/L isopropyl beta-d-thiogalactoside(IPTG)concentration for 4 hours were basically stable.Western blot showed that the purified protein can be recognized by anti-Histag antibody and mouse anti ZZl polyclonal antibody,respectively;The competitive adsorption test showed that the adsorption efficiency of ZZ1 against Acinetobacter baumannii ABo9V decreased by 42.4%compared with 63.5%in the control group in the presence of recombinant ZZlgp12 protein;When both ZZ1 recombinant gp37 protein and ZZ1 recombinant gp12 protein were present,the adsorption ratedecreased by 55.8%.ELISA and further validation assays showed that both ZZl recombinant receptorbinding proteins adsorbed LPS on the surface of the host bacterium ABo9V.Conclusion The recombinant receptorbinding protein gpl2 of the bacteriophage ZZ1 was successfully expr

关 键 词：鲍曼不动杆菌 噬菌体 噬菌体受体结合蛋白 尾丝蛋白 

分 类 号：R378[医药卫生—病原生物学]