人FasL全长cDNA的克隆及其真核表达载体的构建

Molecular Cloning of Full Length Human FasL cDNA and Construction of the Eukaryotic Expression Vector

目的:克隆人FasL全长cDNA并构建其真核表达载体。方法:采用RT鄄PCR法从健康人外周血单核细胞中扩增包含全部阅读框架的FasL全长cDNA,将之与pGEM鄄TEasy质粒连接、测序。构建pcDNA3.1鄄FasL真核表达载体,用脂质体介导方法转染人直肠癌8348细胞,检测FasL表达情况。结果:RT鄄PCR扩增的FasL产物经限制性内切酶酶切后生成的片段符合预期大小。克隆测序证实所得的FasLcDNA序列与GeneBank序列完全一致。pcDNA3.1鄄FasL重组质粒经EcoRI+XhoI双酶切后,电泳显示898bp目的片段和pcDNA3.1载体片段,证明重组质粒正确。转染直肠癌8348细胞后,用RT鄄PCR方法检测FasL表达阳性。结论:采用RT鄄PCR方法成功克隆了人FasL全长cDNA并构建了其真核表达载体,为进一步研究FasL功能奠定了基础。

Objective:To clone FasL cDNA and construct its eukaryotic expression vector.Methods:RT-PCR technique was used to clone the full length of FasL cDNA from actived peripheral mononuclear cells(PBMC)of healthy human donors.Sequencing was performed and the eukaryon expression vector was constructed.Results:The RT-PCR products were digested with restriction endonuclease and the size of the fragement complied with what had been expected.Sequencing result showed the cloned full reading frame of FasL cDNA is in coincidence with the sequence registrated in Gene Bank.The recombinant eukaryotic expression vector of FasL was digested with XhoI and EcoRI,and the electrophoresis showed a898bp fragment and the vector fragment.The recombinant eukaryotic expression vector was transfected into human rectal cancer cell line8348and FasL expression was demonstrated.Conclusion:Human FasL cDNA was cloned and the recombinant eukaryotic expression vector was constructed successfully.