人miR-34a真核表达载体的构建及其表达

Construction of eukaryotic expression vector of human miR-34a and it's expression

目的:构建人miR-34a的真核表达载体,并使其在骨肉瘤SOSP-9607细胞中表达。方法:从人骨肉瘤SOSP-9607细胞基因组DNA中用PCR法扩增出miR-34a的前体序列,并引入酶切位点和保护碱基,双酶切后定向插入真核表达载体pcD-NA3.1(+)中,构建miR-34a真核表达载体pcD-NA-miR34a,然后进行菌落PCR、酶切和测序鉴定。同时用构建好的载体pcDNA-miR34a转染骨肉瘤细胞SOSP-9607细胞,筛选miR-34a稳定表达细胞系,用Northern blot及realtimeRT-RCR法鉴定pcD-NA-miR34a可于真核细胞中过表达miR-34a的生物学特性。结果:成功构建了人miR-34a真核表达载体pcDNA-miR34a。pcDNA-miR34a可于骨肉瘤SOSP-9607细胞中上调miR-34a的表达,获得了miR-34a高表达骨肉瘤细胞系。结论:miR-34a的真核表达载体可在骨肉瘤细胞SOSP-9607细胞中有效表达,为进一步研究miR-34a在骨肉瘤中的功能及基因调控机制奠定了实验基础。

OBJECTIVE：To construct a recombinant eukaryotic expression plasmid of miR-34a and express it in osteosarcoma SOSP-9607 cells. METHODS：miR-34a precursor sequence was amplified by PCR from the human genomic DNA,and then cloned into the pcDNA 3.1（＋） eukaryotic expression vector to produce the recombinant pcDNA-miR34a. The osteosarcoma SOSP-9607 cells were transfected with recombinant pcDNA-miR34a,and then selected to obtain the stable cells,and then the miR-34a expression was verified by Northern blot and real time RT-PCR. RESULTS：The miR-34a eukaryotic expression vector was successfully constructed. pcDNA-miR34a could up-regulate the expression of miR-34a in osteosarcoma SOSP-9607 cells. The miR-34a high expression osteosarcoma cells were obtained. CONCLUSION：The eukaryotic expression plasmid of miR-34a is successfully constructed and effectively expressed in osteosarcoma SOSP-9607 cells,which facilitats the further study of miR-34a fuction in osteosarcoma.