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[Construction of recombinant eukaryotic expression vector pDsRed1-C3/XAPC7 and cellular localization of XAPC7 protein
Jia-yu Tan1, Ya-ling Luo, Xiang Huang
1Central ICU of Boai Hospital of Zhongshan City Affiliated Hospital to Southern Medical University, Zhongshan 528403, China. 28242174@qq.com
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.
