人CD154基因真核细胞表达载体的构建、鉴定及序列分析

Construction of mammalian cell expression vector of human CD154 gene from active peripheral blood mononuclear cell and analysis of its sequence

目的构建人 CD15 4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c DNA中扩增得到 82 0 bp的人 CD15 4c DNA片段 ,再用 Bam H 和 Eco R 双酶切后定向克隆到真核细胞表达载体pc DNA3.1中 ,限制性内切酶酶切分析重组质粒和 DNA序列分析。结果人 CD15 4c DNA已经正确克隆到真核细胞表达载体pc DNA3.1中。结论本研究为探讨 CD15 4分子与淋巴细胞活化的关系及其信号传导机制 ,为进一步用于抗移植排斥反应的研究 ,或对由于 CD15

ObjectiveTo obtain mammalian cell expression vector of human CD154 gene MethodsA 820 bp cDNA fragment was amplified by RT PCR method from 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 1(+) plasmids The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes Bam H Ⅰ and Eco R Ⅰ and sequenced by Sangers dideory mediated chain termination ResultsThis cDNA fragment included 820 bp entire coding region and a part of the 3 non coding region The recombinant mammalian cell expression vector of pcDNA3 1(+)/hCD154 was constructed, the sequence of the insert was identical to the published sequence encoding human CD154 antigen ConclusionThe recombinant mammalian cell expression vector of pcDNA3 1(+)/hCD154 was successfully constructed