类鼻疽伯克霍尔德菌Frataxin样蛋白的表达与纯化  

Expression and purification of the frataxin protein from Burkholderia pseudomallei

作　　者：陈垂这 肖英伦 尹浩燕 吴芸华 张雪连 李肇伟 夏乾峰[1,2,3] CHEN Chui-zhe;XIAO Ying-lun;YIN Hou-yan;WU Yun-hua;ZHANG Xuw-lian;LI Zhaowei;XIA Qian-feng(Ministry of Education Key Laboratory of Tropical Translational Medicine and School of Tropical Medicine and Laboratory Medicine,Hainan Medical University,Haikou,Hainan,China;Faculty of Tropical Medicine and Laboratory Medicine,Hainan Medical College)

机构地区：[1]海南医学院热带医学与检验医学院,海南海口571199 [2]海南医学院热带转化医学教育部重点实验室 [3]海南医学院热带生物医学技术实验室

出　　处：《中国病原生物学杂志》2020年第12期1377-1380,1384,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81960002);科研培育基金项目(No.HYPY201923);校级大学生创新创业训练计划项目(No.X201911810023)。

摘　　要：目的构建重组原核表达质粒,表达类鼻疽伯克霍尔德菌Frataxin样蛋白并进行纯化。方法PCR扩增类鼻疽伯克霍尔德菌Frataxin基因,胶回收纯化后测序验证。用HindⅢ和NdeⅠ双酶切目的片段与pET30a(+),将目的基因片段插入含His标签序列的原核表达载体PET30a(+)中,构建重组表达质粒pET30a(+)-Frataxin,转化入大肠埃希菌BL21(DE3),IPTG诱导目的蛋白的表达,并进行SDS-PAGE和Western blot检测分析,通过His-HP柱及分子筛进行纯化。结果PCR扩增的Frataxin基因片段为327 bp,与预期大小相符。目的片段与原核表达质粒载体连接后进行PCR鉴定与测序验证,重组质粒pET30a(+)-Frataxin构建正确。重组质粒转化DE3后经IPTG诱导5 h,从细菌裂解液中检测到Frataxin融合蛋白,分子质量单位约为12 ku。通过His-HP柱与分子筛纯化,获得单一SDS-PAGE条带的目的蛋白。结论成功表达并纯化了Frataxin样蛋白,为研究Frataxin样蛋白与铁的相互作用关系奠定了基础。Objective To construct a recombinant prokaryotic expression plasmid and to express and purify the frataxin-like protein of Burkholderia pseudomallei.Methods The frataxin gene of B.pseudomallei was amplified using PCR,and the gene was recovered,purified,and sequenced.The gene was digested with two restriction enzymes,Hind III and NdeI,and then ligated into PET30 a(+)containing a His tag sequence.The recombinant plasmid was transformed into E.coli BL21 competent cells(DE3),and expression of the protein was then induced with IPTG.The recombinant protein was analyzed using SDS-PAGE and Western blotting,and it was then purified using His-HP column chromatography.Results Amplification with PCR resulted in a fragment of the frataxin gene that was 327 bp in length,which is consistent with the expected size.The target fragment was ligated into a plasmid vector for prokaryotic expression and then amplified using PCR and sequenced.The recombinant plasmid pET30 a(+)-Frataxin was correctly constructed.The frataxin fusion protein,with a molecular weight of approximately 12 ku,was detected in the bacterial lysate,and it was then purified using an His-HP column and molecular sieve.This yielded the target protein,which produced a single SDS-PAGE band.Conclusion A frataxin-like protein was successfully expressed and purified.This finding has laid the foundation for study of the interaction between frataxin-like proteins and iron.

关 键 词：类鼻疽伯克霍尔德菌 Frataxin样蛋白 原核表达 

分 类 号：R378[医药卫生—病原生物学]