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目的:构建miR-21的真核表达载体,并使其在骨肉瘤MG63细胞中表达,为研究miR-21对骨肉瘤细胞MG63的作用打下基础。方法:根据miRNA的成熟序列以及附近约200多碱基共433个碱基序列,设计PCR引物,PCR扩增,退火后用T4连接到线性化的pcDNA3.1(+)质粒中,并对重组质粒进行双酶切、菌落PCR及测序分析,鉴定完全正确的重组质粒转染骨肉瘤细胞MG63,G418(400 mg/L)筛选4周获得miR-21稳定表达细胞系,用Northern blot及real time RT-RCR法鉴定pcDNA3.1(+)-miR-21可在真核细胞中过表达。结果:成功构建了miR-21的真核表达载体,并获得稳定转染重组质粒的细胞系。结论:miR-21的真核表达载体在骨肉瘤细胞MG63中稳定高表达,为进一步研究miR-21在骨肉瘤MG63中的功能及基因调控机制奠定了实验基础。
Objective: To construct the eukaryotic expression vector of miR-21 and express it in osteosarcoma MG63 cells, which lays the foundation for studying the effect of miR-21 on MG63 cell. Methods: According to the mature sequence of miRNA and a total of 433 base sequences of about 200 bases in the vicinity, a PCR primer was designed and amplified by PCR. After annealed, the PCR product was ligated into the linearized pcDNA3.1 (+) plasmid and recombined The plasmids were double-digested, colony-PCR and sequencing analysis. The correct recombinant plasmids were identified and transfected into osteosarcoma cells MG63 and G418 (400 mg / L) for 4 weeks to obtain miR-21 stably expressing cell lines. Northern blot and real time Identification of pcDNA3.1 (+) - miR-21 by RT-RCR was overexpressed in eukaryotic cells. Results: The eukaryotic expression vector of miR-21 was successfully constructed and the cell line stably transfected with the recombinant plasmids was obtained. CONCLUSION: The eukaryotic expression vector of miR-21 is stably overexpressed in osteosarcoma cell line MG63, which lays the foundation for further studies on the function and gene regulatory mechanism of miR-21 in MG63.