人CD154基因真核表达载体的构建及在Eca109细胞中的表达

Construction of eukaryotic expression vector pcDNA3.1-CD154 and its expression in Eca109 cell line

目的:构建人CD154基因真核表达质粒pcDNA3.1-CD154,初步观察转染该质粒后Eca109细胞的细胞生物学特性。方法:人外周血单个核细胞体外经PHA激活后提取总RNA,用RT-PCR技术扩增人CD154cDNA全长并构建真核表达质粒pcDNA3.1-CD154。用LipofectamineTM2000介导pcDNA3.1-CD154质粒转染食管癌Eca109细胞株,RT-PCR检测转染细胞中CD154mRNA的表达,观察细胞生长特征。结果:用T7/SP6通用引物测序证实重组质粒中插入片段为正向插入的人CD154基因。pcDNA3.1-CD154转染Eca109细胞后细胞异型性明显改善,生长速率减慢。结论:pcDNA3.1-CD154构建成功;转染该质粒的Eca109细胞生物学特性发生改变。

Aim: To construct eukaryotic expression vector pcDNA3.1 GAAB2 CD154 and observe the biological characteristics of Eca109 cells transfected with the recombinant. Methods: Total RNA was extracted from peripheral blood mononuclear cells.The amplification of CD154 cDNA and the construction of PCDNA3.1 GAAB2 CD154 were conducted using RT GAAB2 PCR.Eca109 cell was transfected with PCDNA3.1 GAAB2 CD154,the expression of CD154 mRNA was detected using RT GAAB2 PCR. Results: The inserted sequence in the plasmid pcDNA3.1 GAAB2 CD154 was identfied with human CD154 cDNA sequence provided by GenBank (accession number: NM_000074). The growth velocity of the CD154 gene transfected Eca109 cells decreased and some biological characteristics of the cells altered. Conclusion: pcDNA3.1 GAAB2 CD154 is established successfully,and the characteristics of Eca109 cells have changed after transfecting with the vector.