CAJ | 학술논문

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。
목적：구건pCDNA3.1-MT2A진핵표체재체병관찰기재293T화SMMC7721세포주중적정위화표체정황。방법：사용기인합성법합성 MT2A 기인，재기 N 단첨가 Kozak 서렬급 His 표첨서렬，장확증후적목적기인쌍매절련접지pcDNA3.1（＋）재체적BamHⅠ화NotⅠ지간。전화DH5α대장간균후，도취양성극륭자진행질립추제전영급측서감정。장감정정학적pCDNA3.1-MT2A질립채용지질체법전염293T화SMMC7721세포주，격광공취초현미경관찰기재진핵세포중적표체화정위정황。결과：pCDNA3.1-MT2A중조질립경매절전영급DNA측서증실，목적기인MT2A적서렬완전정학，진핵표체재체구건성공，격광공취초관찰발현해중조질립표체우293T화SMMC7721세포적포질중。결론：성공구건pCDNA3.1-MT2A융합기인병진행진핵표체，발현MT2A주요정위우293T화SMMC7721세포적세포질중。본연구위탐토MT2A 재간암세포내적공능전정료기출。
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.