铁蛋白基因Fth1的克隆及其在大鼠骨髓间充质干细胞中的表达

doi:10.13560/j.cnki.biotech.bull.1985.2017.02.026

生物技术通报 ›› 2017, Vol. 33 ›› Issue (2): 179-184.doi: 10.13560/j.cnki.biotech.bull.1985.2017.02.026

铁蛋白基因Fth1的克隆及其在大鼠骨髓间充质干细胞中的表达

刘伟¹, 朱修良², 李清海²

1. 浙江省农业科学院植物保护与微生物研究所,杭州 310021;
2. 浙江大学医学院附属第二医院放射科,杭州 310009

收稿日期:2016-10-11 出版日期:2017-02-26 发布日期:2017-02-08
作者简介:刘伟,男,博士,研究方向：分子生物学;E-mail：biolwei@sina.com
基金资助:
浙江省自然科学基金项目（LY15H180004）,浙江省公益项目（2015C32020）

Cloning of Ferritin Gene Fth1 and Its Expression in the Bone Marrow Mesenchymal Stem Cells of Rat

LIU Wei¹, ZHU Xiu-liang², LI Qing-hai²

1. Institute of Plant Protection and Microbiology,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021;
2. Department of Radiology,the Second Affiliated Hospital,College of Medical Sciences,Zhejiang University,Hangzhou 310009

Received:2016-10-11 Published:2017-02-26 Online:2017-02-08

摘要/Abstract

摘要： 旨在构建编码大鼠铁蛋白重链多肽1（ferritin heavy chain 1,Fth1）的慢病毒,并检测其在大鼠骨髓间充质干细胞（RMSC）中的表达。PCR扩增大鼠Fth1基因,克隆至pLenti-GFP-C慢病毒表达载体,包装慢病毒,感染RMSC细胞。荧光显微镜观察细胞内GFP荧光情况,Western blot检测Fth1的表达情况,WST-1试剂检测Fth1慢病毒感染组（RMSC-Fth1）和空载体组（RMSC-GFP）的细胞增殖活性。DNA测序结果表明,pLenti-GFP-C-Fth1慢病毒载体构建成功。包装慢病毒感染RMSC细胞后,荧光显微镜下可见明显绿色荧光,表明感染成功。Western blot结果显示,实验组细胞裂解液中可检测到特异的Fth1表达条带。细胞增殖实验显示,与空载体组相比,过表达Fth1并不影响RMSC细胞生长。成功构建并包装携带大鼠Fth1基因的慢病毒,其能够成功感染RMSC细胞并表达Fth1蛋白,且对细胞增殖无明显影响。

关键词: Fth1, 磁共振报告基因, 慢病毒载体, 大鼠骨髓间充质干细胞

Abstract: Aimsare to construct a lentivirus encoding rat ferritin heavy chain 1（Fth1）and to detect its expression in rat bone mesenchymal stem cells（RMSCs）. The rat Fth1 gene was amplified by PCR and cloned into the lentivirus expression vector pLenti-GFP-C. The lentivirus carrying pLenti-GFP-C-Fth1 or empty vector was then packaged in HEK 293T cells,by which the RMSCs were infected. GFP expression was observed under a fluorescence microscope. Fth1 expression was detected by Western blot. WST-1 reagent was used to analyze the proliferation of infected cells（RMSC-Fth1）and empty vector（RMSC-GFP）. Resultsof DNA sequencing showed that the pLenti-GFP-C-Fth1 lentivirus expression vector was successfully constructed. After RMSCs were infected by the packaged lentivirus,GFP was observed under a fluorescence microscope,indicating the infection was achieved. A specific band of Fth1 in the lysate of Fth1-lentivirus infected cells（RMSC-Fth1）was detected by Western blot. Cell proliferation assay showed that the growth of RMSC was not affected by overexpressing Fth1 compared with RMSC-GFP. As conclusion,the recombinant lentivirus vector carrying rat Fth1gene was successfully constructed,infected RMSC as expected,and expressed FTH1 protein even without significant impacts on the cell proliferation.

Key words: Fth1, MRI reporter, lentivirus vector, rat bone mesenchymal stem cell

刘伟, 朱修良, 李清海. 铁蛋白基因Fth1的克隆及其在大鼠骨髓间充质干细胞中的表达[J]. 生物技术通报, 2017, 33(2): 179-184.

LIU Wei, ZHU Xiu-liang, LI Qing-hai. Cloning of Ferritin Gene Fth1 and Its Expression in the Bone Marrow Mesenchymal Stem Cells of Rat[J]. Biotechnology Bulletin, 2017, 33(2): 179-184.

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铁蛋白基因Fth1的克隆及其在大鼠骨髓间充质干细胞中的表达

Cloning of Ferritin Gene Fth1 and Its Expression in the Bone Marrow Mesenchymal Stem Cells of Rat

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