[Construction of recombinant eukaryotic expression vector pDsRed1-C3/LOC51255 and subcellular localization of LOC51255 protein in HePG2 cell line].
OBJECTIVE
To construct the recombinant eukaryotic expression vector pDsRed1-C3/LOC51255 and investigate the subcellular localization of LOC51255 protein in HePG2 cell line.


METHODS
The cDNA of LOC51255 was amplified from the recombinant plasmid pET28b/LOC51255 by PCR. The aim gene fragment LOC51255 from pMD18-T/LOC51255 was subcloned into eukaryotic expression vector pDsRed1-C3. The recombinant plasmid pDsRed1-C3/LOC51255 was identificated by BamH I/Xho I double digestion and sequence analysis, and then transfected into HePG2 cell line by lipofectamine 2000. The expression of LOC51255 protein and its subcellular localization were detected by fluorescence microscope.


RESULTS
The construction of the recombinant plasmid pDsRed1-C3/LOC51255 was proved by restriction enzyme digestion analysis and DNA sequencing. Its red fluorescent protein was mainly detected in the cytoplasm of HePG2 cell line.


CONCLUSIONS
The eukaryotic expression plasmid pDsRed1-C3/LOC51255 has been successfully constructed and expressed in the HepG2 cell line. LOC51255 protein has been proved to be located in the cytoplasm of HePG2 cell line. Our research will help further studies on the function of human gene LOC51255.
Department of Etiology, Medical College of Jiaying University, Meizhou, China. xiaotaoqil1998@126.com
Xiang
X
Huang
Jia-yu
JY
Tan
Yu-lin
YL
Qiu
Wen-jun
WJ
Liang
Ming
M
Zhou
Ming
M
Li
D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D044767:Ubiquitin-Protein Ligases; D000097573:Red Fluorescent Protein; C547603:RNF181 protein, human