Construction of a fusion protein expression vector pGS/2SS-M4GFP without antibiotic resistance gene and its subcellular localization in different cell lines
Construction of a fusion protein expression vector pGS/2SS-M4GFP without antibiotic resistance gene and its subcellular localization in different cell lines
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无抗生素抗性基因融合蛋白表达载体pGS/2SS-M4GFP的构建及其在不同细胞系中的亚细胞定位
DOI：
10.1016/j.biologicals.2008.09.002
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发表时间：
2009-01-01
期刊：
影响因子：
1.7
通讯作者：
Yang, L. G.
中科院分区：
文献类型：
--
作者：
Liang, A. X.;Han, L.;Yang, L. G.
本研究通过将GS/2SS基因与增强型绿色荧光蛋白(M4GFP)序列重组，构建了新的表达载体pGS/2SS-M4GFP。首次将编码两个拷贝的生长抑素基因的GS/2SS融合基因导入pVAX-ASD载体，用ASD盒取代卡那霉素抗性盒。然后将M4GFP基因融合到GS/2SS基因的3‘端。纯化后，将pGS/2SS-M4GFP分别导入猪肾癌细胞和人癌细胞。RT-PCR证实GS/2SS基因的转录过程，共聚焦显微镜和ELISA法观察GS/2SS-M4GFP融合蛋白的定位和表达。结果表明，单核M4GFP定位于胞浆和胞核，而融合蛋白GS/2SS-M4GFP仅定位于胞浆。此外，应该注意的是，GS/2SS-M4GFP的亚细胞定位不是某一细胞系所特有的，而是在各种细胞系中似乎是共同的。这些结果首次为M4GFP是一种示踪GS/2SS蛋白的通用工具提供了有价值的证据，并为进一步研究其在体内的组织分布和免疫机制奠定了基础。(C)2008年国际生物制品协会。爱思唯尔有限公司出版。保留所有权利。
A novel plasmid pGS/2SS-M4GFP was constructed in the present study by recombination of GS/2SS gene and enhanced green fluorescent protein (M4GFP) sequence. The GS/2SS fusion gene encoding two copies of somatostatin genes was firstly introduced into pVAX-asd vector in which the kanamycin resistance cassette was replaced by the asd cassette. The M4GFP gene was then fused into 3' end of GS/2SS gene in the proper reading frame. After purified, plasmid pGS/2SS-M4GFP was transfected into different cell lines derived from pig kidney and human cancer cells. The transcription process of GS/2SS gene was confirmed by RT-PCR, and the localization as well as expression of GS/2SS-M4GFP fusion protein was observed by confocal microscopy and ELISA. Transfection results revealed that sole M4GFP was localized within the cytosol and the nucleus, while fusion protein GS/2SS-M4GFP was localized only in the cytoplasm. Furthermore, it should be noted that subcellular localization of GS/2SS-M4GFP was not specific to one cell line, but appeared to be common across a variety of cell lines. These results provide for the first time valuable evidence that M4GFP is a versatile tool to trace GS/2SS protein and pave the way for further study on its tissue distribution and immunological mechanism in vivo. (c) 2008 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.