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目的构建表达小鼠新基因mgt-16的反转录病毒载体,并观察其在小鼠胚胎间充质干细胞C3H/10T1/2(简称10T1/2细胞)中的表达。方法以含小鼠新基因mgt-16的DNA序列为模板PCR扩增得到mgt-16编码序列,T-A克隆后测序获得pMD18T-16质粒,与真核表达载体pEGFP-N1酶切、连接、转化,通过PCR、酶切鉴定和测序获得正确的pEGFP-N1-16载体。将pEGFP-N1-16载体中含mgt-16的片段克隆至反转录病毒载体pLEGFP-N1,通过酶切鉴定和测序获得正确的pLEGFP-N1-16反转录病毒载体。将pLEGFP-N1-16转染反转录病毒包装细胞Phoenix,制备携带mgt-16与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的反转录病毒,感染小鼠间充质干细胞10T1/2后,400μg/mL G418筛选获得稳定表达mgt-16与EGFP融合蛋白的10T1/2细胞克隆。荧光显微镜观察MGT-16蛋白的表达及亚细胞定位。结果 PCR扩增得到大小约300bp的mgt-16条带,T-A克隆后测序显示获得的mgt-16序列与Genbank数据库序列相同。构建的pEGFP-N1-16载体经PCR、酶切鉴定和测序验证构建成功,构建的反转录病毒载体pLEGFP-N1-16经酶切鉴定和测序验证构建成功。荧光显微镜观察MGT-16主要在Phoenix细胞和小鼠间充质干细胞的细胞质表达,核周表达水平较高。结论成功构建了小鼠新基因mgt-16的反转录病毒载体,并在间充质干细胞中表达,为进一步研究新基因mgt-16在间充质干细胞中的功能奠定了基础。
Objective To construct retroviral vector expressing murine gene mgt-16 and observe its expression in mouse embryonic mesenchymal stem cells C3H / 10T1 / 2 (10T1 / 2 cells for short). Methods The mgt-16 coding sequence was amplified by PCR using the DNA sequence containing mouse novel gene mgt-16. After TA cloning and sequencing, the plasmid pMD18T-16 was obtained and digested, ligated and transformed with eukaryotic expression vector pEGFP-N1. The correct pEGFP-N1-16 vector was obtained by PCR, restriction enzyme digestion and sequencing. The fragment containing mgt-16 in pEGFP-N1-16 vector was cloned into the retroviral vector pLEGFP-N1, and the correct pLEGFP-N1-16 retroviral vector was obtained by restriction analysis and sequencing. The pLEGFP-N1-16 was transfected into the retrovirus packaging cell Phoenix to prepare a retrovirus carrying mgt-16 and enhanced green fluorescent protein (EGFP) and infected with mouse mesenchymal stem cells 10T1 / 2, 400 μg / mL G418 screened to obtain 10T1 / 2 cell clone stably expressing mgt-16 and EGFP fusion protein. The expression of MGT-16 protein and subcellular localization were observed by fluorescence microscopy. Results A band of about 300bp of mgt-16 was obtained by PCR. Sequencing of T-A clone showed that the obtained mgt-16 sequence was the same as the GenBank database. The constructed pEGFP-N1-16 vector was successfully constructed by PCR, restriction enzyme digestion and sequencing. The constructed retroviral vector pLEGFP-N1-16 was successfully constructed by restriction enzyme digestion and sequencing. The expression of MGT-16 was mainly observed in the cytoplasm of Phoenix cells and mouse mesenchymal stem cells by fluorescence microscopy. Conclusion The mouse retroviral vector containing mgt-16 gene was successfully constructed and expressed in mesenchymal stem cells, which laid the foundation for further study of the function of mgt-16 in mesenchymal stem cells.