类鼻疽伯克霍尔德菌AraC转录因子基因的原核表达及纯化  

Prokaryotic expression and purification of AraC transcription factor from Burkholderia pseudomallei

作　　者：曾慧[1] 黄玮晨 高洁 江昱颖 胡琪 修皓 ZENG Hui;HUANG Wei-chen;GAO Jie;JIANG Yu-ying;HU Qi;XIU Hao(School of Tropical Medicine,HaiNan Medical University,Haikou 571199,China)

机构地区：[1]海南医学院热带医学院,海南海口571199

出　　处：《中国病原生物学杂志》2022年第7期761-765,共5页Journal of Pathogen Biology

基　　金：海南省自然科学基金项目(No.819QN236);海南医学院科研培育基金项目(No.HYPY201904);海南医学院大学生创新创业训练计划项目(No.X202011810024)。

摘　　要：目的从海南类鼻疽伯克霍尔德菌菌株BPHN001中克隆得到AraC转录因子基因,构建类鼻疽伯克霍尔德菌(Burkholderia pseudomallei)AraC转录因子基因原核表达质粒,对该转录因子基因进行原核表达,并对表达产物进行纯化。方法根据AraC转录因子基因设计特异性扩增引物,将PET-28a(+)质粒和扩增得到的AraC转录因子基因产物同时用BamHⅠ和HindⅢ双酶切,构建带His标签的重组质粒。经测序验证后,将重组质粒转化至大肠埃希菌BL21(DE3),采用最佳浓度IPTG进行诱导,采用SDS-PAGE鉴定重组蛋白的表达情况。通过Ni^(2+)柱纯化目的蛋白,采用Western blot检测纯化蛋白的免疫反应性。结果特异性引物扩增得到的产物大小约为1000 bp,与目的基因片段1032 bp基本一致。将扩增产物与PET-28a(+)载体连接后测序,与预期完全一致。重组载体转化BL21(DE3)后用最适IPTG浓度(0.5 mmol/L)诱导过夜(约16 h),SDS-PAGE检测表达产物分子质量在40~55 ku之间,与预期相符。表达蛋白纯化后进行Western blot,能被相应抗体识别。结论成功克隆得到AraC转录因子基因,,构建的含AraC转录因子基因原核表达载体转化DE3后经IPTG诱导可表达出融合蛋白,经Ni^(2+)柱纯化得到具有免疫反应原性的AraC转录因子蛋白,为AraC转录因子的功能研究奠定了基础。Objective Cloned a new AraC transcription factor from BPHB001 which was isolated from HaiNan.Constructed the prokaryotic expression plasmid of AraC transcription factor,expressed and purified the AraC transcription factor protein in prokaryotic expression system.Methods The AraC transcription factor gene was cloned and verified by polymerase chain reaction(PCR)and sequencing.The recombination vector pET28 a(+)-AraC contain His-taq was constructed and verified by PCR and enzyme digestion(BamHⅠand HindⅢ).The recombination vector pET28 a(+)-AraC was transformed into Escherichia coli BL21(DE3).The expression of fusion protein was induced with Optimum IPTG concentration and detected with SDS-PAGE and purified with Ni^(2+)chelate column.Then the purification products was identified by Western blot.Results A fragment was amplified using specific primer,the size of the fragment was bigger than 1000 bp which was consistent with expectation(1032 bp).The recombination vector pET28 a(+)-AraC was verified by sequencing.The E.coli BL21(DE3)strain containing pET28 a(+)-AraC vector was induced with IPTG(0.5 mmol/L)and cultured for overnight(almost 16 hours).The expression of fusion protein was detected by SDS-PAGE,and verified by western blot assay.Conclusion An AraC transcription factor gene was cloned and a recombination vector carrying AraC gene was successfully constructed.The target protein was successfully expressed in E.coli BL21(DE3)by IPTG inducing.The fusion AraC transcription factor protein with Immunoreactivity was purified by using Ni^(2+)chelate column.These results will lay the foundation for further function study on AraC transcription factor.

关 键 词：类鼻疽伯克霍尔德菌 AraC转录因子 原核表达 

分 类 号：R378[医药卫生—病原生物学]