人BLyS基因真核细胞表达载体的构建和鉴定

Construction of mammalian cell expression vector of human BLyS gene from activated peripheral blood mononuclear cell and analysis of its sequence

目的 构建人BLyS基因真核细胞表达载体。方法 采用RT PCR方法 ,从激活的人外周血淋巴细胞的总cDNA中扩增得到 876bp的人BLyScDNA片段 ,再用XhoⅠ和EcoRⅠ双酶切后定向克隆到真核细胞表达载体pcDNA3中 ,用限制性内切酶酶切分析和DNA序列分析鉴定重组质粒。结果 人BLyScDNA已经正确克隆到真核细胞表达载体pcDNA3中。

Objective To obtain mammalian cell expression vector of human BLyS gene. Methods In this report, a 876 bp cDNA fragment was amplified by RT PCR method from the total RNA of human peripheral blood mononuclear cell(PBMC) activated with 10 ng/mL PMA and 1 μg/mL PHA for 8 hours. The fragment was cloned into pcDNA3 plasmids. The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes Xho Ⅰ and Eco R Ⅰ and sequenced by Sanger dideoxy mediated chain termination. Results The results showed that this cDNA fragment included 876 bp entire coding region. The recombinant mammalian cell expression vector of pcDNA3/hBLyS was constructed, the sequence of the insert was identical to the published sequence encoding human BLyS antigen. Conclusion The recombinant mammalian cell expression vector of pcDNA3/hBLyS was successfully constructed.