【摘要】 目的:克隆小鼠B7-DC基因,并构建含该基因的真核表达载体pEF6/-B7-DC-V5His,证明该基因在CHO细胞中的表达方式,为建立B7-DC的转基因小鼠打下基础。方法:从小鼠胸腺中抽提总RNA为模板,通过RT-PCR技术,扩增出B7-DC编码区片段。按常规方法克隆进pEF6V5His载体中,重组质粒经鉴定后采用脂质体法转染CHO细胞,48小时后进行间接免疫荧光实验,72小时后用blasticidin进行筛选,挑选出能稳定表达B7-DC的细胞株。结果:已成功克隆小鼠B7-DC基因并构建了用于表达B7-DC基因的真核表达载体,经间接免疫荧光实验(IFA)证明,该基因在CHO细胞中得到表达。经Western blot检测表明筛选出的转基因细胞稳定表达了B7-DC基因。结论:构建了用于表达B7-DC基因的真核表达载体pEF6/-B7-DC-V5His,并能在CHO细胞中稳定表达目的基因,为该基因功能的后续研究奠定了基础。更多还原

【Abstract】 Objective:To clone human B7-DC gene and construct recombinant eukaryotic expression vector pEF6/-B7-DC-V5His carrying the target gene.Methods:Total RNA was isolated from mouse thymus and subjected to reverse transcription, and then the mouse B7-DC gene was amplified by RT-PCR. The PCR product was cloned into T vector and the sequence was confirmed by restriction enzyme digestion and sequencing. Then the specific B7-DC encoding region fragment was subcloned into the eukaryotic expression vector pEF6/V5His A. The recombinant plasmid was transfected into CHO cells with liposome transfection reagent for transient expression.CHO cell line stably expressing B7-DC was selected in the presence of blasticidin.Results:The coding sequence of B7-DC gene was successfully colned, and recombinant eukaryotic expression vector for expressing B7-DC was constructed. The target protein was detected by cellular immunofluorescence technology.Conclusion:Construction of recombinant eukaryotic expression vector pEF6/-B7-DC-V5His which can express B7-DC gene stably in CHO cells could contribute to further biological function research preparation.更多还原