P268S突变型NOD2/CARD15真核表达载体的构建及其体外表达

Construction of P268S Mutant NOD2/CARD15 Eukaryotic Expression Vector and its Expression in vitro

背景:NOD2/CARD15基因序列单核苷酸多态性(SNP)与欧美人群的克罗恩病(CD)明显相关,其中R702W、G908R和3020insC3个SNP位点与CD的相关性尤为显著。而日本、韩国以及我国香港和浙江地区的研究均未发现上述3个SNP的改变,但最近研究发现了可能与中国人CD相关的P268S突变。目的:构建P268S突变型NOD2/CARD15真核表达载体和体外转染体系,为研究突变型NOD2/CARD15的功能提供实验基础。方法:应用定点诱变技术构建P268S突变型NOD2/CARD15真核表达载体,以阳离子脂质体介导体外转染技术瞬时转染人胚肾细胞HEK293T,以蛋白质印迹法和逆转录聚合酶链反应(RT-PCR)检测HEK293T细胞NOD2/CARD15的表达。结果:经克隆、酶切、测序证实获得P268S突变型NOD2/CARD15基因,突变载体转入HEK293T细胞后,NOD2/CARD15有效表达。结论:成功构建了P268S突变型NOD2/CARD15真核表达载体,阳离子脂质体是人胚肾细胞有效的体外转染体系。

Background： The single nucleotide polymorphism （SNP） of NOD2/CARD15 gene is associated with Crohn＇s disease （CD） in European and American population, especially the three SNP R702W, G908R and 3020insC, whereas not in Japanese, Korean and people in Hongkong, Zhejiang area in China. But SNP P268S mutation was found to be associated with Chinese CD patients recently. Aims： To construct P268S mutant NOD2/CARD15 eukaryotic expression vector and transfection system in vitro, providing an experimental basis for further study on the function of mutant NOD2/CARD15. Methods： The site mutation was induced by in vitro site-directed mutagenesis, HEK293T cells were transfected by LipofectamineTM 2000. The expression of NOD2/CARD15 was detected by western blot and reverse transcriptase polymerase chain reaction （RT-PCR）. Results： The mutant plasmid was correctly constructed, NOD2/CARD15 was expressed after the mutant plasmid was transfected in HEK293T cells. Conclusions： The constructed eukaryotic expression plasmid （pcDNANOD2-P268S-HA） could express NOD2/CARD15 in HEK293T cells in vitro.