【摘要】 目的构建鼠CD40配体(Mcd40)cDNA真核表达载体,为进一步研究打下基础。方法从鼠脾细胞中提取总RNA作为模板,用特异的引物和RT-PCR法扩增mCD40L cDNA。PCR产物T-A克隆了T载体构建中间重组体,NheI和EcoRI双酶切后,将Mcd40L cDNA定向插入真核表达载体pcDNA3.1多克隆位点中,构建成重组表达质粒,并用酶切分析、PCR扩增和序列测定进行了鉴定。结果mCD40L cDNA被正确地克隆到真核表达载体pcDNA3.1+中,测序结果同文献报道的序列一致。结论真核表达载体pcDNA3.1+-mCD40L的成功构建,为开展利用mCD40L基因治疗肿瘤的研究奠定了基础。更多还原

【Abstract】 Objective: To construct an eukaryotic expression vector carrying the murine CD40 ligand cDNA (mCD40L cDNA) as a basis for further study. Methods: Cellular total RNA was extracted from murine splenocytes as a template. The mCD40L cDNA was synthesized by RT-PCR with the specific primers. The mCD40L cDNA fragment directly cloned into T vector to generate middle recombinant. After restriction endonuclease NheI and EcoRI double digested it, the target fragment was subcloned into eukaryotic vector pcDNA3.1+ to construct eukaryotic expression recombinant. The recombinant plasmid was verified with restriction analysis and sequencing. Results: The mCD40L cDNA was cloned correctly into vector pcDNA3.1+. The resultant sequence was completely consistent with the published sequence. Conclusion: The eukaryotic expression vector pcDNA3.1+-mCD40L is constructed successfully making it possible to study further on gene therapy tumors with mCD40L gene.更多还原