人TLT-2真核表达载体的构建及其在L929细胞株中的稳定转染

Construction of Eukaryotic Expressing Vector of Human TLT-2 and Establishment of Stable Transfectant L929 Cell Line

目的构建含有人TLT-2基因的真核表达载体,并通过转染获得稳定表达TLT-2分子的L929转基因细胞株。方法运用逆转录聚合酶链式反应(RT-PCR)技术从健康人外周血单个核细胞(PBMC)的cDNA文库获得全长TLT-2基因,经过双酶切(XhoI和EcoRI)装入逆转录病毒表达载体pIRES2-EGFP中,脂质体法转染L929细胞,经G418抗性筛选,流式细胞术、RT-PCR及Western blot鉴定TLT-2分子的表达。结果构建了真核表达载体pIRES2-EGFP/hTLT-2,建立了稳定转染TLT-2目的基因的L929细胞株。结论成功构建了TLT-2真核表达载体并获得了稳定转染该分子的转基因细胞,为进一步研究人TLT-2分子的生物学功能提供了物质基础。

Objective To construct eukaryotic expressing vector of human trem-like transcript 2（TLT-2） gene and transfect L929 cells so as to establish stable L929 cell line.Methods cDNA fragment encoding TLT-2 was obtained from human lymphocyte cells by reverse transcription.The TLT-2 gene was then designed with the restiction endonucleases XhoI and EcoRI and inserted into corresponding region of pIRES2-EGFP vector.And then the recombinant pIRES2-EGFP/TLT-2 was transfected into L929 cells by lipofectanime TM2000.After screening culture by G418,stable transfected L929 cell line was established,and the expression of TLT-2 was identified by FCM,RT-PCR and Western blot.Results The eukaryotic expressing vector pIRES2-EGFP/TLT-2 was constructed,stable transfected L929 cell line was established,and TLT-2 gene was expressed successfully.Conclusion The construction of eukaryotic expressing vector pIRES2-EGFP/TLT-2 and the development of stable transfected L929 cells have provided a solid experiment foundation for further studies of TLT-2 fuction.