人CD154基因绿色荧光真核表达载体的构建

Construction of Human CD154 Gene Eukaryotic Green Fluorescent Protein Expression Vector pEGFP-N3-CD154

目的构建携人CD154基因的重组真核绿色荧光蛋白表达载体pEGFP N3-CD154。方法体外PHA激活人外周血单个核细胞,提取总RNA,用RT-PCR技术扩增人CD154 cDNA全长开放阅读框序列,T-A克隆至pGEM_TEasy载体,再定向克隆至增强型绿色荧光蛋白载体pEGFP-N3,重组质粒pEGFP_N3_CD154用限制性内切酶酶切鉴定,通用鉴定引物PCR扩增鉴定,并进行DNA序列分析。结果多种鉴定方法证实了重组载体中的CD154基因序列与已知的序列一致。结论成功构建了携带人CD154基因的重组的增强型绿色荧光蛋白表达载体pEGFP-N3-CD154,为进一步研究人CD154基因转染细胞在肿瘤免疫治疗中的生物学意义提供初步的实验基础。

Objective To construct recombined eukaryotic green fluorescent protein expression vector pEGFP-N3-CD154.Methods Total RNA was extracted from peripheral blood mononuclear cells （PBMCs） which were activated with PHA in vitro. The amplification of CD154 cDNA open reading frame sequence was conducted using PT-PCR. The fragment of CD154 cDNA was cloned into pGEM T Easy vector, and then cloned directly into pEGFP-N3. The recombinant plasmid pEGFP-N3-CD154 was identified by restriction enzyme, PCR,and DNA sequence analyzing technique. Results CD154 gene sequence in the recombined vector was identical with the GenBank （NM-000074）. Conclusion The recombined eukaryotic green fluorescent protein expression vector pEGFP-N3-CD154 can be obtained. The results provide basis for the further studies of the biological significance of human CD154 gene transfected cell in tumor immunotherapy.