鼠CD40配体cDNA真核表达载体的构建

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—PCIL法扩增mCD40L cDNA。PCIL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、PCIL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。

Construction of recombined murine CD40 ligand cDNA eukaryotic expression vector

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.