摘要
本研究旨在构建人TEFM基因的shRNA真核表达载体,对其抑制效率进行鉴定,以筛选出抑制效率最优的shRNA干扰载体.根据人TEFM基因全长cDNA序列(NM_024683.3),利用Ambion公司在线软件设计出4条shRNA,分别克隆至psi-U6载体中,得到4个重组干扰质粒TEFM-shRNA-1~TEFM-shRNA-4.重组子转化感受态细胞后挑取阳性克隆子进行PCR鉴定和DNA测序验证.将4个shRNA真核表达载体分别转染人脑胶质瘤U87细胞,使用嘌呤霉素筛选稳定转染细胞株后,通过qPCR和Western blotting分别检测各组细胞中TEFM的mRNA和蛋白质表达水平.PCR鉴定和DNA测序鉴定证实重组质粒中已插入目的DNA序列.qRT-PCR和Western blotting结果表明,4个重组载体均可以显著降低TEFM的mRNA和蛋白质表达水平(P<0.01),其中以TEFM-shRNA-3和TEFM-shRNA-4的抑制效率最优,对TEFM mRNA表达的抑制率分别为76.5%和78.8%,TEFM蛋白表达的抑制率分别为69.5%和74.8%.成功构建TEFM基因shRNA真核表达载体,转染人脑胶质瘤U87细胞并成功筛选出稳定沉默细胞株,为进一步探索TEFM在脑胶质瘤中的生物学功能和作用机制奠定了实验基础.
Human mitochondrial transcription elongation factor(TEFM)is a molecular switch for mitochondrial gene replication and transcription conversion and get involved in the occurrence and development of malignant tumors.The purpose of this study is to construct shRNA eukaryotic expression vectors of human TEFM gene,and to identify their inhibition efficiency,so as to screen out the shRNA interference vector with the best inhibition efficiency.According to the full-length cDNA sequence of human TEFM(NM_024683.3),four shRNA target sequences were designed with Ambion’s online software,and four complementary oligonucleotide strands were cloned into psi-U6 vector.Among them,four recombinant interference plasmids TEFM-shRNA-1~TEFM-shRNA-4 were obtained.After transforming competent cells,positive clones are picked for PCR identification and DNA sequencing.Four shRNA eukaryotic expression vectors were transfected into human glioma U87 cells,and the mRNA and protein expression of TEFM gene in each group were detected by qRT-PCR and Western blotting.Through the above methods,the most effective shRNA interference plasmid was screened out.PCR identification and DNA sequencing confirmed that the target DNA sequence has been inserted into the recombinant plasmid.qRT-PCR and Western blotting results showed that the four recombinant vectors can significantly reduce the expression levels of TEFM mRNA and protein(P<0.01).The inhibition efficiency of TEFM-shRNA-3 and TEFM-shRNA-4 was the best,the inhibition rate of TEFM mRNA expression was 76.5%and 78.8%,respectively.The inhibition rate of TEFM protein expression was 69.5%and 74.8%,respectively.In Conclusion,the shRNA eukaryotic expression vector of TEFM gene was successfully constructed,transfected into human glioma U87 cells,and a stable silent cell line was successfully screened.The results will provide experimental basis for further study on the biological function and mechanism of TEFM in brain glioma.
作者
刘如爱
飞再镱
李彬
杨玺
王播勇
余敏
熊伟
LIU Ruai;FEI Zaiyi;LI Bin;YANG Xi;WANG Boyong;YU Min;XIONG Wei(School of Basic Medical Sciences,Dali University,Dali,Yunnan 671000,China;Key Laboratory of Clinical Biochemistry of Yunnan Province,Dali University,Dali,Yunnan 671000,China;School of Life Sciences,Yunnan University,Kunming,Yunnan 650091,China)
出处
《平顶山学院学报》
2022年第5期117-123,共7页
Journal of Pingdingshan University
基金
国家自然科学基金项目(31760331,32160167,82160516)
云南省李云庆专家工作站(202005AF150014)
云南省万人计划青年拔尖人才(2019)
云南省高校病理学与病理生理学硕士研究生导师团队项目(2019)
云南省教育厅科研基金研究生项目(2020Y0542)
国家级大学生创新创业训练计划项目(202010679014,202010679027)。
