艰难梭菌CysE和CysK蛋白的克隆、表达与生物信息学分析  

Clonal expression and bioinformatics analysis of CysE and CysK proteins of Clostridioides dif ficile

作　　者：古文鹏[1] 贾森泉 周永明[1] 尹建雯[1] 赵世文[1] 赵晓南[1] 伏晓庆[1] GU Wenpeng;JIA Senquan;ZHOU Yongming;YIN Jianwen;ZHAO Shiwen;ZHAO Xiaonan;FU Xiaoqing(Department of Acute Infectious Disease Control and Prevention,Yunnan Provincial Center for Disease Control and Prevention,Kunming 650500,China)

机构地区：[1]云南省疾病预防控制中心急性传染病防制所,云南昆明650500

出　　处：《中国病原生物学杂志》2024年第11期1266-1270,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.82360398);云南省基础研究专项项目(No.202301AT070160);云南省技术创新人才培养对象项目(No.202405AD350003)。

摘　　要：目的生物信息学分析艰难梭菌半胱氨酸合成蛋白CysE和CysK的理化性质、三维结构和蛋白互作信息,并进行两个蛋白的体外克隆表达和纯化。方法采用ProtParam对CysE和CysK蛋白进行理化性质预测,TMHMM进行跨膜结构域分析,SignalP分析蛋白信号肽,SWISS-MODEL预测二者的三维结构,STRING分析蛋白互作信息。将cysE和cysK目的基因片段分别克隆到pET-32a和pET-28a载体,融合His标签通过大肠埃希菌表达系统来表达目的蛋白,并进行表达纯化测试。结果CysE蛋白共有196氨基酸(AA),无跨膜区,信号肽序列为无,疏水性一般,有2个无序性片段。CysK蛋白共有302 AA,无跨膜区,信号肽序列为无,疏水性一般,有5个无序性片段。两个蛋白共产生α螺旋、β片层、β转角和无规卷曲4种二级结构。CysE的二级结构共由5个α螺旋、15个β片层、10个β转角和19个无规卷曲组成;CysK的二级结构共由15个α螺旋、16个β片层、16个β转角和26个无规卷曲组成。蛋白互作预测结果显示,CysE与CysK涉及菌株的蛋白合成和代谢途径,主要是半胱氨酸的合成途径、甲硫氨酸的合成以及硫代谢途径等。重组表达载体pET-32a-cysE和pET-28a-cysK构建成功,对蛋白诱导表达条件进行优化后,进行SDS-PAGE电泳。结果显示,两个蛋白的分子量分别为CysE 38 ku和CysK 34 ku,二者的纯度大于85%。最终得到的CysE重组蛋白浓度为0.8 mg/mL,CysK重组蛋白的浓度为1 mg/mL。结论本研究对艰难梭菌CysE和CysK的蛋白进行了生物信息学分析,并成功构建和表达了CysE与CysK重组蛋白,为进一步探索二者对于艰难梭菌致病性的相关机制提供了基础。Objective The physicochemical properties,three-dimensional structures and protein interactions of the Clostridioides difficile cysteine synthesis proteins CysE and CysK were analyzed,and these two proteins were cloned,expressed and purified.Methods Physicochemical properties of CysE and CysK proteins were predicted using ProtParam,transmembrane structural domainswere analyzed by TMHMM,protein signal peptide were analyzed by SignalP,three-dimensional structure of proteins were predicted by SWISS-MODEL,and protein interactions information were analyzed by STRING.The target gene cysE and cysK were cloned into pET-32a and pET-28a vectors,respectively,fused with His-tags to express the target proteins through the E.coli expression system,and then tested for expression purification.Results The CysE protein had a total of 196 amino acids(AA),no transmembrane region,a signal peptide sequence of nil,fair hydrophobicity,and two disordered fragments.The CysK protein had a total of 302 AA,no transmembrane region,a signal peptide sequence of nil,fair hydrophobicity,and five disordered fragments.The two proteins showed a total of 4 secondary structures ofα-helices,β-sheet layers,β-turns and random curls.The secondary structure of CysE consisted of a total of 5α-helices,15β-sheet layers,10β-turns,and 19 random curls;the CysK consisted of a total of 15α-helices,16β-sheet layers,16β-turns,and 26 random curls.The protein interactions prediction showed that CysE and CysK were involved in protein synthesis and metabolic pathways of the strain,mainly referred to cysteine synthesis pathway,methionine synthesis,and sulfur metabolism pathway.The recombinant expression vectors pET-32a-cysE and pET-28a-cysK were successfully constructed,and SDS-PAGE electrophoresis was carried out after the optimization of protein-induced expression conditions.The results showed that the molecular weights of the two proteins were 38 ku for CysE,34 ku for CysK,respectively,and the purity of the two was greater than 85%.The final concentration of C

关 键 词：艰难梭菌 CysE CysK 克隆表达 生信分析 

分 类 号：R378[医药卫生—病原生物学]