人Fn14基因真核重组质粒的构建及其表达

目的构建人成纤维细胞生长因子诱导早期反应蛋白14（fibroblast growth factor—inducible 14，Fn14）基因真核重组质粒及其表达。方法从人肝细胞癌组织抽取总RNA，RT—PCR扩增出Fn14基因全长cDNA，经测序正确后，核酸内切酶EcoR I、Xba I酶切PCR产物，亚克隆人pcDNA3．1-Myc载体，重组质粒pcDNA3．1-Myc—Fn14经酶切及测序鉴定正确后转染COS-7细胞，通过免疫组织化学DAB法检测Myc—Fn14融合蛋白的表达。结果重组质粒pcDNA3．1-Myc—Fn14经EcoR I、Xba I酶切及测序鉴定正确，重组质粒包含人Fn14基因完整编码区。免疫组织化学检测结果显示，重组质粒转染COS-7细胞后成功表达Myc—Fn14融合蛋白，转染空载体pcD—NA3．1-Myc的COS-7细胞未见Myc—Fn14融合蛋白表达。结论构建了人Fn14基因真核表达载体，该重组质粒可表达含Myc-Fn14的融合蛋白，为研究Fn14功能奠定了基础。

Construction of Recombinant Eukaryotic Expression Vector of Human Fn14 Gene and Expression

Objective To construct the recombinant eukaryotic expression vector of human Fn14 gene and to examine the express of the Fn14 protein in COS-7 cell line. Methods Total RNA was isolated from human hepatoma tissue by Trizol DAB method. The full-length of Fn14 cDNA was amplified by using RT-PCR method. The amplification products were cloned in the EcoR I/Xba I cut pcDNA 3.1-Myc vector and sequenced. The Myc-Fn14 fusion protein was detected by immunohistochemical DAB method after COS-7 cell line transfected by the recombinant pcDNA 3.1-Myc-Fn14 vector. Results The recombinant plasmid pcDNA 3. 1-Myc-Fn14 was digested with EcoR I/Xba I . Its full-length Fn14 cDNA fragment subcloned into plasmid pcDNA 3.1-Myc vector was confirmed by sequenced analysis. The COS-7 cell line transfection by the recombinant pcDNA 3.1-Myc-Fn14 expressed the Myc-Fn14 fusion protein was detected by immunohistochemical method. However the Myc-Fn14 fusion protein was not detected in COS-7 cell line transfected by the pcDNA 3. 1-Myc vector. Conclusion The successful construction of expression vector of Fn14 gene and its protein expression in COS-7 cells line lays the foundation on the further study on the function of Fn14 protein.