肺炎克雷伯菌氨基肽酶A的克隆表达与活性鉴定  

Cloning,expression and activity identification of aminopeptidase peptidase A from Klebsiella pneumoniae

作　　者：白丽民 张筱薇 李笑眉 龚政 汪佳希 韩雨佳 徐刚[1,2] Bai Limin;Zhang Xiaowei;Li Xiaomei;Gong Zheng;Wang Jiaxi;Han Yujia;Xu Gang(Department of Burns and Plastic Surgery,Northern Jiangsu People’s Hospital,Yangzhou 225001,China;Medical College of Yangzhou University,Yangzhou 225001,China;Graduate School of Dalian Medical University,Dalian 116044,China)

机构地区：[1]苏北人民医院烧伤整形科,扬州225001 [2]扬州大学临床医学院,扬州225001 [3]大连医科大学研究生院,大连116044

出　　处：《中华实验外科杂志》2024年第4期697-699,共3页Chinese Journal of Experimental Surgery

摘　　要：目的对肺炎克雷伯菌(KP)中标注的氨基肽酶(PepA)进行序列扩增与蛋白表达,鉴定其酶活特性。方法根据(美国)国家生物技术信息中心(NCBI)公布的KP PepA基因序列(Reference Sequence:NZ_CP045783.1)设计扩增引物,以本科室前期分离到的一株KP基因组为模板,通过聚合酶链反应(PCR)扩增出PepA基因全长,使用NdeⅠ和XhoⅠ双酶切后连接至表达载体pET-30a。将重组质粒pET-30a-PepA转化至大肠杆菌BL21(DE3),经异丙基硫代半乳糖苷(IPTG)诱导表达出KP PepA蛋白,使用N^(2+)层析柱对重组His标签蛋白进行纯化。以亮氨酸-对硝基苯胺(Leu-pNA)为底物,测定KP PepA对其水解能力,并摸索不同的反应温度和pH值条件对水解反应的影响。在反应体系中加入不同二价金属离子,观察各离子对KP PepA氨基肽酶活性的作用。组间比较采用t检验。结果扩增出长度为1527 bp的KP PepA基因,并获得可溶性KP PepA蛋白,大小为56.1×103,与预测大小一致。KP PepA加入组反应产物pNA浓度显著高于KP PepA不加组[(205.00±5.00)μmol/L比(8.00±2.00)μmol/L,t=63.36,P<0.01],表明重组KP PepA可以水解Leu-pNA。KP PepA水解Leu-pNA的最适温度为37℃,最适pH值为pH 8.0。Co^(2+)、Mn^(2+)均可以提高KP PepA酶活性,其中Co^(2+)激活作用最强,加入Co^(2+)组的相对酶活性显著高于不加离子组(181.70±7.64比95.00±0.00,t=16.44,P<0.01);Zn^(2+)、Fe^(2+)均可以抑制KP PepA酶活性,其中Zn^(2+)抑制作用最强,加入Zn^(2+)组的相对酶活性显著低于不加离子组(44.67±8.97比95.00±0.00,t=8.49,P<0.01)。结论肺炎克雷伯菌中标注的PepA具有体外氨基肽酶活性。Objective To amplify and prokaryotically express a probable peptidase A(PepA)from Klebsiella pneumoniae(KP),and identify its enzymatic activity characteristics.Methods According to the KP PepA sequence published by the(US)National Center for Biotechnology Information(NCBI),the PepA open reading frame(ORF)was amplified by polymerase chain reaction(PCR),then cloned into the pET-30a vector,and transformed into Escherichia coli BL21(DE3),induced by isopropylβ-D-thiogalactoside(IPTG),and the recombinant protein rPepA was obtained after Ni+chromatography gel purification.Using Leucine-p-Nitroaniline(Leu-pNA)as the substrate,the hydrolysis ability of KP PepA was determined,and the effects of different reaction temperatures and pH conditions on the hydrolysis reaction were explored.Different divalent metal ions were added to the reaction system to observe the effect of each ion on the activity of KP PepA aminopeptidase.The t test was used for comparison between groups.Results The KP PepA gene with a length of 1527 bp was amplified,and the soluble recombinant KP PepA protein was obtained with a size of 56.1×103,consistent with the predicted sizes.The pNA concentration(reaction product)in the group with KP PepA addition was significantly higher than that in the group without KP PepA[(205.00±5.00)vs.(8.00±2.00)μmol/L,t=63.36,P<0.01],indicating that KP PepA can hydrolyze Leu-pNA.The optimal temperature for hydrolyzing Leu-pNA by KP PepA was 37℃,and the optimal pH value was 8.0.Both Co2+and Mn2+can increase the activity of KP PepA enzyme,among which Co2+had the strongest activation effect.The relative enzyme activity of the group adding Co2+was significantly higher than that of the group without metal ions(181.70±7.64 vs.95.00±0.00,t=16.44,P<0.01);Zn2+and Fe2+can inhibit KP PepA enzyme activity,among which Zn2+had the strongest inhibitory effect.The relative enzyme activity of the group adding Zn2+was significantly lower than that of the group without metal ions(44.67±8.97 vs.95.00±0.00,t=8.49,P<0.01).Conclusion Pe

关 键 词：肺炎克雷伯菌 氨基肽酶 亮氨酸 对硝基苯胺 

分 类 号：R563.1[医药卫生—呼吸系统]