摘要
目的对类鼻疽伯克霍尔德菌TssE-6蛋白进行原核表达和蛋白纯化并进行生物信息学分析。方法采用BamHI和HindⅢ双酶切PCR纯化产物和pET30a(+)载体后连接,构建pET30a(+)-tssE-6重组质粒,转化到BL21大肠埃希菌中,利用1 mmol/L ITPG诱导,获得目的蛋白,进行10%SDS-PAGE、Western blot验证及Ni;柱纯化,利用生物学信息学软件对TssE-6蛋白进行预测和分析。结果成功对TssE-6蛋白进行了原核表达,经10%SDS-PAGE和Western blot验证,包含组氨酸标签的蛋白相对分子质量为24.2×10^(3)。TssE-6为不可溶包涵体蛋白,Ni;柱纯化蛋白的浓度为3.0 mg/ml,目的条带单一。TssE-6蛋白为含有170个氨基酸的不稳定亲水蛋白,带负电荷,分子式C_(851)H_(1349)N_(235)O_(255)S_(7),理论相对分子质量为19.176 91×10^(3),二级结构中α螺旋和无规卷曲分别占46.47%和40.00%。结论获得高纯度的重组TssE-6蛋白,预测该蛋白具有亲水性,为进一步研究TssE-6在T6SS中的功能以及新的检测标志物奠定了理论基础。
Objective To express the TssE-6 protein of Burkholderia pseudomallei in E.coli,to purify that protein,and to bioinformatically analyze that protein.Methods After double restriction endonuclease digestion of tssE-6 with BamHI and HindⅢ,the purified PCR product was ligated into a pET30 a(+)vector to construct the recombinant plasmid pET30 a(+)-tssE-6,which was then transformed into E.coli BL21.Expression of the target protein was successfully induced with 1 mmol/L ITPG,and the protein was verified as correct using 10%SDS-PAGE and Western blotting.After assessing its solubility,the TssE-6 protein was purified on an Ni^(2+)column.Bioinformatic software was used to predict and analyze the TssE-6 protein.Results The recombinant plasmid pET30 a(+)-tssE-6 was successfully constructed,and TssE-6 protein was successfully expressed in E.coli.The molecular weight of the protein containing a histidine tag was 24.2×10^(3),which was verified using 10%SDS-PAGE and Western blotting.Solubility analysis indicated that TssE-6 was an insoluble inclusion body protein.The Ni^(2+) column yielded the purified protein in a concentration of 3.0 mg/mL,and the protein produced a single target band.Bioinformatic analysis indicated that the TssE-6 protein consisting of 170 amino acids was an unstable hydrophilic protein with a negative charge.Its molecular formula was C_(851)H_(1349)N_(235)O_(255)S_(7) and its relative molecular weight was 19.17691×10^(3).α-helices accounted for 46.47%of the secondary structure of the TssE-6 protein and random coils accounted for 40.00%.Conclusion This study yielded highly pure TssE-6 protein and it revealed information about its basic characteristics.These findings will provide a theoretical foundation for further study of the function of TssE-6 in T6 SS and its use as a new marker.
作者
董素芳
谭晶晶
刘成利
费鹏
夏乾峰
DONF Su-fang;TAN Jing-jing;LIU Cheng-li;FEI Peng;XIA Qiang-feng(School of Tropical Medicine and Laboratory Medicine,Laboratory of Tropical Biomedicine and Biotechnology,Hainan Medical University,Haikou,Hainan 571199,China;Clinical Laboratory of Wuhan Hospital of Traditional Chinese Medicine,Wuhan,Hubei 430010,China)
出处
《中国病原生物学杂志》
CSCD
北大核心
2021年第10期1163-1167,共5页
Journal of Pathogen Biology
基金
海南省自然科学基金项目(No.817138)
海南省科协青年科技英才创新计划项目(No.QCXM201701)
大学生创新创业训练计划项目(No.HYCX2018014)。
