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目的:构建过表达母系表达印记基因MEG3(materally expressed gene 3,MEG3)全长的慢病毒载体,观察其对骨髓瘤细胞凋亡的影响。方法:以包含MEG3全长的包装质粒pcDNA3.0-MEG3为模板,设计针对MEG3全长的引物(寡核苷酸片段),经PCR扩增后获得MEG3全长,并将其亚克隆入线性化的慢病毒表达载体pCDH-EF1-copGFP中,经双酶切、PCR及测序等方法鉴定。利用脂质体转染试剂将鉴定正确的pCDH-EF1-M EG3-copGFP重组质粒转染HEK-293T细胞,进一步通过荧光显微镜和流式细胞术检测GFP表达效率,real-time PCR检测MEG3mRNA表达水平。通过三质粒共转染及PEG纯化的方法获得慢病毒,以优化感染滴度感染骨髓瘤细胞系,通过流式细胞术检测过表达MEG3对细胞凋亡的影响。结果:经酶切、PCR及测序等方法证实成功构建pCDH-EF1-M EG3-copGFP过表达载体;通过流式细胞术测定制备的慢病毒滴度为1.86×10~8pfu/ml;流式细胞术检测和realtime PCR方法证实MEG3在293T细胞和骨髓瘤细胞中表达明显上调;流式细胞术检测证实过表达MEG3可诱导骨髓瘤细胞凋亡。结论:成功构建并制备过表达MEG3的慢病毒过表达载体pCDH-EF1-MEG3-copGFP,该载体能高效过表达MEG3并诱导骨髓瘤细胞凋亡,进一步证实了MEG3具有抑癌功能,为进一步研究MEG3调控骨髓瘤细胞生物学特性及机制奠定了基础。
OBJECTIVE: To construct a lentiviral vector that overexpresses the materally expressed gene 3 (MEG3) and observe its effects on the apoptosis of myeloma cells. Methods: The full-length MEG3 primer (oligonucleotide fragment) was designed based on the plasmid pcDNA3.0-MEG3 containing the full length of MEG3. The full length of MEG3 was amplified by PCR and subcloned into the linear The lentiviral expression vector pCDH-EF1-copGFP was identified by double enzyme digestion, PCR and sequencing. The recombinant plasmid pCDH-EF1-MEG3-copGFP was transfected into HEK-293T cells by liposome transfection reagent. The efficiency of GFP expression was detected by fluorescence microscope and flow cytometry. The expression of MEG3 mRNA was detected by real-time PCR . The lentivirus was obtained by co-transfection of three plasmids and purified by PEG, and the myeloma cell line was infected with the optimized infection titer. The effect of overexpression of MEG3 on apoptosis was detected by flow cytometry. Results: The recombinant plasmid pCDH-EF1-M EG3-copGFP was successfully constructed by restriction enzyme digestion, PCR and sequencing. The titer of lentivirus was 1.86 × 10-8pfu / ml by flow cytometry. Flow cytometry The expression of MEG3 in 293T cells and myeloma cells was detected by both real-time PCR and real-time PCR. Flow cytometry confirmed that MEG3 overexpression induced myeloma cell apoptosis. Conclusion: MEG3 overexpression vector pCDH-EF1-MEG3-copGFP was successfully constructed and prepared, which can efficiently overexpress MEG3 and induce apoptosis of myeloma cells, further confirming that MEG3 has a tumor suppressor function. For further study MEG3 regulation of myeloma cell biological characteristics and mechanisms laid the foundation.