pCDNA3.1-MT2A真核表达载体的构建及亚细胞定位

Construction of recombinant eukaryotic expression vector pCDNA3. 1-MT2A and cellular localization of MT2A protein in different cell lines

目的:构建pCDNA3.1-MT2A真核表达载体并观察其在293T和SMMC7721细胞株中的定位和表达情况。方法:使用基因合成法合成MT2A基因,在其N端添加Kozak序列及His标签序列,将扩增后的目的基因双酶切连接至pcDNA3.1(+)载体的BamHⅠ和NotⅠ之间。转化DH5α大肠杆菌后,挑取阳性克隆子进行质粒抽提电泳及测序鉴定。将鉴定正确的pCDNA3.1-MT2A质粒采用脂质体法转染293T和SMMC7721细胞株,激光共聚焦显微镜观察其在真核细胞中的表达和定位情况。结果:pCDNA3.1-MT2A重组质粒经酶切电泳及DNA测序证实,目的基因MT2A的序列完全正确,真核表达载体构建成功,激光共聚焦观察发现该重组质粒表达于293T和SMMC7721细胞的胞质中。结论:成功构建pCDNA3.1-MT2A融合基因并进行真核表达,发现MT2A主要定位于293T和SMMC7721细胞的细胞质中。本研究为探讨MT2A在肝癌细胞内的功能奠定了基础。

Objective： To construct the recombinant eukaryotic expression vector pCDNA3.1-MT2 A and to investigate the cellular localization of MT2 A protein in 293 T and SMCC7721 cell lines. Methods： Gene synthesis method was used to synthetic gene MT2 A,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-MT2 A plasmids which passed through electrophoretic and sequenced were transfected 293 T 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-MT2 A was confirmed by restriction analysis and DNA sequencing,the sequence of the target gene MT2 A 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-MT2 A and expressed in eukaryotic cells,we found that the MT2 A was mainly localized in the cytoplasm of 293 T and SMMC7721 cell lines. The findings can help us to lay the foundation for the functions of MT2 A in hepatoma cells.