| 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，400 μg/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300 bp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。 |
| 关键词: 新基因 过表达 逆转录病毒 间充质干细胞 |
| DOI：10.3724/SP.J.1008.2014.00447 |
| 投稿时间：2013-08-28修订日期：2013-10-08 |
| 基金项目:国家自然科学基金（31201035）；国家重点基础研究发展计划（973计划，2005CB522605）；全军医学科技“十二五”重大项目（AWS11C004）. |
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| Construction of retrovirol vector containing novel gene, mgt-16, and its expression in mouse mesenchymal stem cells |
| 王明科 |
| () |
| Abstract: |
| Objective To construct the retroviral vector carrying novel gene mgt-16 and to investigate its expression in mouse mesenchymal stem cells C3H/10T1/2(10T1/2). Methods DNA sequences containing mouse novel gene mgt-16 was used as a template for PCR amplification of full length mgt-16 cDNA. Then the DNA fragment was cloned into pEGFP-N1 vector to produce pEGFP-N1-16 vector after T-A cloning with pMD18T plasmid and sequencing. The pEGFP-N1-16 vector 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-N1-16 vector including mgt-16 gene. The pLEGFP-N1-16 vector was verified by restriction enzyme digestion, sequenced, and then transfected into packaging cell line Phoenix to prepare EGFP fused mgt-16 retrovirus particles, which were collected and used to infect 10T1/2 cells. G418 (400 μg/mL) continuous selection was conducted to obtain 10T1/2 cell clones stably overexpressing EGFP fused mgt-16. Fluorescence microscope was employed to determine the expression and subcellular localization of MGT-16 in Phoenix and 10T1/2 cells. Results A band of about 300 bp size was observed by agarose gel electrophoresis after PCR amplification for mgt-16 gene, and the result of sequencing showed that the sequence of insert fragment in T-A clones was identical to mgt-16 gene reported in Genbank. PCR, restriction enzyme digestion and sequencing revealed that the pEGFP-N1-16 plasmid was successfully constructed. Restriction enzyme digestion and sequencing revealed that the pLEGFP-N1-16 plasmid was also successfully constructed. Phoenix and 10T1/2 cells overexpressing MGT-16 showed 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-16 in mesenchymal stem cells. |
| Key words: novel gene over-expression retrovirus mesenchymal stem cells |
