摘要
目的构建RECK(reversion-inducing cysteine-rich protein with kazal motifs)基因mRNA 3′非编码区(3′-untranslated region,3′UTR)全长及两个截短片段的荧光素酶报告基因载体,以进一步研究microRNA对RECK基因的调控。方法以人乳腺癌细胞系MCF-7基因组DNA为模板,PCR扩增RECK基因mRNA的3′UTR全长及两个截短片段,利用双酶切将目的片段定向插入报告基因载体pGL3-Control中,将克隆载体转化大肠埃希菌DH5α感受态细胞进行扩增,采用菌落PCR、双酶切、测序鉴定重组子,双荧光报告基因检测其表达活性。结果重组载体中RECK3′UTR序列与GenBank序列比对一致且插入方向正确;3个重组载体分别命名为pGL3-RECK 3′UTR-1、pGL3-RECK3′UTR-2和pGL3-RECK 3′UTR-3,转染细胞后可表达荧光素酶基因。结论成功构建了RECK基因3′UTR全长及两个截短片段的荧光素酶报告基因载体,为进一步研究microRNA对靶基因RECK的调控奠定了基础。
Objective To construct the luciferase reporter gene vector for full length and two truncated fragments of RECK 3′UTR so as to further study the regulation of microRNA to RECK gene. Methods The full length and two truncated fragments of RECK mRNA were amplified by PCR using the genomic DNA of human breast cancer MCF-7 cells as template, digested with two restriction enzymes and inserted into luciferase reporter vector pGL3-Control. The constructed recombinant plasmids were transformed to competent E. coli DH5α. The recombinants were identified by colony PCR,enzyme digestion and sequencing,while the luciferase activity was determined by reporter gene assay.Results The RECK 3′UTR sequences in recombinant plasmids named as pGL3-RECK 3′UTR-1,pGL3-RECK 3′UTR-2 and pGL3-RECK 3′ UTR-3 respectively were consistent with those in Gen Bank,and were inserted correctly. Luciferase gene was expressed in the cells transfected by all the recombinant plasmids. Conclusion The luciferase reporter gene vector for full length and two truncated fragments of RECK 3′UTR was constructed correctly,which laid a foundation of further study on RECK gene regulation through micro RNAs.
作者
伊丽娜
刘睿
张烜
昌妍希
孙鹏
YI Li-na , LIU Rui, ZHANG Xuan, CHANG Yan-xi, SUN Peng(College of Phormacy, Inner Mongolia Medical University, Hohhot 010100, Inner Mongolia Autonomous Region, Chin)
出处
《中国生物制品学杂志》
CAS
CSCD
2018年第3期247-250,256,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金项目"基于转录组学研究肿瘤转移中骨桥蛋白对肿瘤细胞释放外泌体的调控"(81660482)
内蒙古医科大学科技百万工程项目"靶向RECK基因的microRNA对乳腺癌转移调控机制研究"(YKD2015KJBW005)
