Construction of recombinant eukaryotic expression vector pCDNA3.1-MT2A and cellular localization of MT2A protein in different cell lines / 中国免疫学杂志

ABSTRACT

Objective:To construct the recombinant eukaryotic expression vector pCDNA3.1-MT2A and to investigate the cellular localization of MT2A protein in 293T and SMCC7721cell lines.Methods: Gene synthesis method was used to synthetic gene MT2A,added a Kozak sequence and His tag sequence at the N-terminus,the amplified target gene was connected to the pcDNA3.1(+) vector which was double digested between the BamH Ⅰ and Not Ⅰ.After transformation to E.coli DH5α, the positive clones were picked for plasmid extraction then Electrophoretic and sequenced.The pCDNA3.1-MT2A plasmids which passed through electrophoretic and sequenced were transfected 293T and SMMC7721 cell lines by liposome method,and then observed their expression and localization in eukaryotic cells by laser confocal microscopy.Results: The recombinant plasmid pCDNA3.1-MT2A was confirmed by restriction analysis and DNA sequencing,the sequence of the target gene MT2A was entirely correct,eukaryotic expression vector was successfully constructed and cell lines which had transfected recombinants could see the expression of green fluorescent protein in the cytoplasm.Conclusion:Successfully constructed fusion gene of pCDNA3.1-MT2A and expressed in eukaryotic cells,we found that the MT2A was mainly localized in the cytoplasm of 293T and SMMC7721 cell lines.The findings can help us to lay the foundation for the functions of MT2A in hepatoma cells.