摘要
目的构建带有His标签的人乳酸脱氢酶A(LDHA)基因原核表达载体,并进行蛋白纯化,为研究LDHA蛋白功能奠定基础。方法以人乳腺文库作为模板,通过PCR技术扩增得到LDHA基因的编码序列,插入到pET-28a(+)载体中并测序。LDHA在大肠杆菌中进行诱导表达并纯化蛋白,SDS-PAGE及Western印迹检测蛋白纯化效果。采用His pull-down技术检测纯化蛋白与沉默信息调节因子2(SIRT2)的相互作用。结果PCR扩增获得长度约1000 bp的LDHA编码序列,并插入到pET-28a(+)载体中,序列测定结果显示重组质粒构建成功。SDS-PAGE及Western印迹结果显示获得带有His标签的LDHA蛋白。His pull-down实验证明His-LDHA能与SIRT2体外结合,证实其生物学活性良好。结论成功构建带His标签的LDHA基因原核表达载体,并得到纯化蛋白,为进一步研究LDHA蛋白的功能奠定了基础。
Objective To construct a prokaryotic expression vector of human lactate dehydrogenase A(LDHA)gene with His-tag and purify the protein.Methods The CDS sequence of human LDHA was amplified from the human mammary gland library by PCR,and then inserted into the pET-28 a(+)vector,and sequencing was used to confirm.LDHA was induced mildly,and the purified protein was detected by SDS-PAGE and Western blotting.The interaction between purified proteins and Sirtuins 2(SIRT2)was detected by His pull-down technique.Results LDHA gene with a length of about 1000 bp was obtained by PCR and inserted into the pET-28 a(+)vector.pET-28 a(+)-LDHA vector was obtained.LDHA protein with His-tag was purified.His pull-down assay showed that His-LDHA could interact with SIRT2,which verified its known function.Conclusion The pET-28 a(+)-LDHA prokaryotic expression vector has been constructed,and LDHA protein with His tag was purified.The study lays a foundation for further research on the function of LDHA protein.
作者
葛祥伟
陈静漪
石烟祝
蒋琦炜
米月
谢天
叶棋浓
汪进良
GE Xiang⁃wei;CHEN Jing⁃yi;SHI Yan⁃zhu;JIANG Qi⁃wei;MI Yue;XIE Tian;YE Qi⁃nong;WANG Jin⁃liang(Medical School of Chinese PLA,Beijing 100853,China;Department of Oncology,the Fifth Medical Center,Chinese PLA General Hospital,Beijing 100071,China;Department of Cell Engineering,Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Medical College,Guizhou University,Guiyang 550025,China)
出处
《军事医学》
CAS
2021年第10期726-730,共5页
Military Medical Sciences
基金
国家自然科学基金(81930078)
