摘要
目的构建带有绿色荧光蛋白(GFP)标签的人乳酸脱氢酶A(LDHA)的真核表达载体,并对其生物学功能进行初步研究。方法利用PCR技术从人乳腺文库中扩增出人LDHA基因,并将其克隆到pEGFP-C1载体上,经双酶切和测序鉴定成功后转染到肝癌HepG2细胞中,通过Western印迹检测其表达情况,免疫荧光检测其亚细胞定位,并利用生长曲线、克隆形成以及划痕实验探究LDHA对肝癌HepG2细胞增殖、迁移能力的影响。结果从人乳腺文库中扩增获得大小约为1000 bp的DNA片段,并成功克隆至pEGFP-C1载体上,经测序与目的序列完全一致;转染肝癌HepG2细胞后目的基因成功表达;荧光显微镜观察发现,GFP-LDHA融合蛋白主要聚集于细胞质中;细胞生长曲线和克隆形成实验结果显示,转染pEGFP-LDHA的肝癌细胞较空载体细胞增殖能力增强;划痕实验显示,转染pEGFP-LDHA的肝癌细胞较空载体细胞迁移性增强。结论成功构建了带GFP标签的人LDHA真核表达载体,为进一步研究LDHA在肝癌细胞中的功能奠定了基础。
Objective To construct a eukaryotic expression vector of human lactate dehydrogenase A(LDHA)labeled with GFP tag and detect its activity.Methods The human LDHA gene was obtained from the human mammary cDNA library by PCR and cloned into the pEGFP-C1 vector.Human HepG2 cells were transfected with the recombinant plasmid pEGFP-LDHA,whose expression was detected by Western blotting.The subcellular localization of GFP-LDHA was detected by immunofluorescence.The proliferative and migratory capacity of HepG2 cells with LDHA overexpression was examined.Results An LDHA eukaryotic expression vector labeled with GFP tag was successfully constructed by double digestion identification and sequencing.The expression of pEGFP-LDHA in human HepG2 cells was identified by Western blotting.Immunofluorescence showed that LDHA protein was mainly concentrated in the cytoplasm.In addition,human LDHA protein could promote the proliferation and migration of HepG2 cells significantly.Conclusion A eukaryotic expression vector pEGFP-LDHA is successfully constructed,which can be of help for further study of the role of LDHA in the development and progression of hepatocellular carcinoma.
作者
张德宇
马路园
朱祥
刘娟
杨旭辉
陈玉
徐小洁
叶棋浓
ZHANG De-yu;MA Lu-yan;ZHU Xiang;LIU Juan;YANG Xu-hui;CHEN yu;XU Xiao-jie;YE Qi-nong(Department of Medical Molecular Biology,Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Department of Respiration,Xuanwu Hospital,Capital Medical University,Beijing 100053,China)
出处
《军事医学》
CAS
北大核心
2019年第6期406-410,共5页
Military Medical Sciences
基金
国家自然科学基金（81822037,81672589）
