新基因mgt-16反转录病毒载体的构建及其在小鼠间充质干细胞中的表达  

Construction of retrovirol vector containing novel gene mgt-16 and its expression in mouse mesenchymal stem cells

作　　者：王明科[1,2,3] 孙慧勤[2] 程晋[4] 姜帆[4] 粟永萍[2] 邹仲敏[4] 

机构地区：[1]海军医学研究所舰艇卫生研究室,上海200433 [2]第三军医大学军事预防医学院全军复合伤研究所,创伤,烧伤与复合伤国家重点实验室,重庆400038 [3]解放军441医院防疫科,福鼎355200 [4]第三军医大学军事预防医学院毒理学研究所,重庆400038

出　　处：《第二军医大学学报》2014年第4期447-451,共5页Academic Journal of Second Military Medical University

基　　金：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)~~

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。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 cell

关 键 词：mgt-16 过表达 反转录病毒 间质干细胞 

分 类 号：R349.83[医药卫生—基础医学]