【摘要】 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。更多还原

【Abstract】 Objective To construct the retroviral vector carrying novel gene mgt-16and to investigate its expression in mouse mesenchymal stem cells C3H/10T1/2(10T1/2).Methods DNA sequences containing mouse novel gene mgt-16was used as a template for PCR amplification of full length mgt-16cDNA.Then the DNA fragment was cloned into pEGFP-N1vector to produce pEGFP-N1-16vector after T-A cloning with pMD18Tplasmid and sequencing.The pEGFP-N1-16vector was confirmed by PCR,restriction enzyme digestion and sequencing analysis.The retroviral vector,pLEGFP-N1-16,was constructed using retroviral vector,pLEGFP-N1,and pEGFP-N1-16vector including mgt-16gene.The pLEGFPN1-16vector was verified by restriction enzyme digestion,sequenced,and then transfected into packaging cell line Phoenix to prepare EGFP fused mgt-16retrovirus particles,which were collected and used to infect 10T1/2cells.G418(400μg/mL)continuous selection was conducted to obtain 10T1/2cell clones stably overexpressing EGFP fused mgt-16.Fluorescence microscope was employed to determine the expression and subcellular localization of MGT-16in Phoenix and 10T1/2cells.Results A band of about 300bp size was observed by agarose gel electrophoresis after PCR amplification for mgt-16gene,and the result of sequencing showed that the sequence of insert fragment in T-A clones was identical to mgt-16gene reported in Genbank.PCR,restriction enzyme digestion and sequencing revealed that the pEGFP-N1-16plasmid was successfully constructed.Restriction enzyme digestion and sequencing revealed that the pLEGFP-N1-16plasmid was also successfully constructed.Phoenix and 10T1/2cells overexpressing MGT-16showed green fluorescence distribution in the cytoplasmic,especially around the perinuclear area.Conclusion We have successfully constructed a recombinant retroviral vector carrying novel gene,mgt-16,and expressed it in mouse mesenchymal stem cells,which provides a basis for studying the role of novel gene mgt-16in mesenchymal stem cells.更多还原