CAJ | 학술논문

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.
목적구건서CD40배체(Mcd40)cDNA진핵표체재체,위진일보연구타하기출.방법종서비세포중제취총RNA작위모판,용특이적인물화RT-PCR법확증mCD40L cDNA.PCR산물T-A극륭료T재체구건중간중조체,NheI화EcoRI쌍매절후,장Mcd40L cDNA정향삽입진핵표체재체pcDNA3.1다극륭위점중,구건성중조표체질립,병용매절분석、PCR확증화서렬측정진행료감정.결과mCD40L cDNA피정학지극륭도진핵표체재체pcDNA3.1+중,측서결과동문헌보도적서렬일치.결론진핵표체재체pcDNA3.1+-mCD40L적성공구건,위개전이용mCD40L기인치료종류적연구전정료기출.
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.