pDsRed1-C3/XAPC7真核表达载体的构建及细胞定位

Construction of recombinant eukaryotic expression vector pDsRed1-C3/XAPC7 and cellular localization of XAPC7 protein in different cell lines

目的:构建pDsRed1-C3/XAPC7真核表达载体并观察其在786-O、293T和Chang liver细胞株中的定位情况。方法:采用RT-PCR法从HBE135-E6E7细胞株中克隆得到XAPC7cDNA全长序列,将之与pMD18T载体连接、测序后将该片段亚克隆到真核表达载体pDsRed1-C3中。构建好的pD-sRed1-C3/XAPC7真核表达质粒经酶切和测序鉴定后,采用脂质体法将该重组质粒转染786-O、293T和Chang liver细胞株中,观察其在真核细胞中的表达。结果:pDsRed1-C3/XAPC7重组子经酶切鉴定及DNA测序证实,目的基因XAPC7的序列完全正确,真核表达载体构建成功,转染该重组子的细胞株均可见红色荧光蛋白的表达,呈颗粒状分布。XAPC7主要定位于786-O细胞的胞质,胞核罕见,在293T细胞中,胞质和胞核均有分布,且两者间无明显差别,在Changliver细胞中,胞质和胞核亦均有分布,但以胞质为主。结论:成功构建pDsRed1-C3/XAPC7融合基因并进行真核表达,发现XAPC7在不同细胞中的细胞定位具有差异,为下一步研究XAPC7的肿瘤相关功能奠定基础。

AIM： To construct the recombinant eukaryotic expression vector pDsRed1-C3/XAPC7 and to investigate the cellular localization of XAPC7 protein in 786-O and 293T and Chang liver cell lines.METHODS： The cDNA of XAPC7 was amplified from HBE135-E6E7 cell line by RT-PCR method.The aim gene fragment XAPC7 from pMD18-T/XAPC7 was subcloned into eukaryotic expression vector pDsRed1-C3.The recombinant plasmid pDsRed1-C3/XAPC7 was identificated by BamH I/Xho I double digestion and sequence analysis,and then transfected into786-O and 293T and Chang liver cell lines by lipofectamine 2000.The expression and cellular localization of XAPC7 protein were detected by fluorescence microscope.RESULTS： The construction of the recombinant plasmid pDsRed1-C3/XAPC7 was proved successfully by restriction enzyme digestion analysis and DNA sequencing.Red fluorescent protein pDsRed1-C3/XAPC7 was distributed granularly in transfected cell lines.Our researches showed that XAPC7 protein was mainly expressed in the cytoplasm of 786-O cell line,rare in its nucleus,evenly distributed in the cytoplasm and nucleus of 293T cell line,and mainly located in the cytoplasm of Chang liver cell line,few in its nucleus.CONCLUSION： The eukaryotic expression plasmid pDsRed1-C3/XAPC7 has been successfully constructed and expressed in the 786-O and 293T and Chang liver cell lines,but there was obvious difference in different cell lines.Our researches will help further studies on the function of human gene XAPC7.