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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。
Objective To construct eukaryotic expression vector of mouse CD40 ligand (Mcd40) cDNA and lay the foundation for further study. Methods Total RNA was extracted from murine splenocytes and used as template to amplify mCD40L cDNA with specific primers and RT-PCR. The PCR product TA was cloned into the T vector to construct the intermediate recombinants. After double digestion with NheI and EcoRI, the Mcd40L cDNA was inserted into the eukaryotic expression vector pcDNA3.1 multiple cloning site to construct a recombinant expression plasmid. The recombinant plasmid was digested with restriction enzyme digestion, PCR Amplification and sequencing were identified. Results The mCD40L cDNA was correctly cloned into the eukaryotic expression vector pcDNA3.1 +. The sequencing result was consistent with that reported in the literature. Conclusion The successful construction of eukaryotic expression vector pcDNA3.1 + -mCD40L laid the foundation for the research of using mCD40L gene to treat tumors.