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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。
OBJECTIVE: To construct the eukaryotic expression vector pDsRed1-C3 / XAPC7 and observe its localization in 786-O, 293T and Chang liver cell lines. Methods: The full length of XAPC7 cDNA was cloned by RT-PCR from HBE135-E6E7 cell line and ligated with pMD18T vector. After sequencing, the fragment was subcloned into eukaryotic expression vector pDsRed1-C3. The constructed eukaryotic expression plasmid pD-sRed1-C3 / XAPC7 was identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transfected into 786-O, 293T and Chang liver cell lines by lipofectamine and observed in eukaryotic Expression in cells. Results: The recombinant plasmid pDsRed1-C3 / XAPC7 was confirmed by restriction enzyme digestion and DNA sequencing. The sequence of the target gene XAPC7 was completely correct and the eukaryotic expression vector was constructed successfully. The expression of red fluorescent protein was observed in the transfected cells Granular distribution. XAPC7 mainly located in the cytoplasm of 786-O cells, with rare nuclei. In 293T cells, the cytoplasm and nucleus were distributed with no significant difference between the two. In the Changliver cells, the cytoplasm and nuclei There are distribution, but mainly cytoplasm. Conclusion: The pDsRed1-C3 / XAPC7 fusion gene was successfully constructed and expressed in eukaryotic cells. It was found that the cell localization of XAPC7 in different cells was different, which laid the foundation for the further study on the tumor-related function of XAPC7.