人鞘磷脂合酶干扰载体的构建与鉴定  

Construction and identification of RNA interference vector of human sphingomyelin synthase

作　　者：胡时栋[1] 丁毅[1] 宋丹丹[1] 陈清兰[1] 颜念龙[1] 

机构地区：[1]南昌大学基础医学院生物化学与分子生物学教研室,南昌330006

出　　处：《复旦学报（医学版）》2013年第5期584-588,共5页Fudan University Journal of Medical Sciences

基　　金：南昌大学2012年本科创新实验项目(2012184)~~

摘　　要：目的构建沉默人鞘磷脂合酶1(SMS1)和鞘磷脂合酶2(SMS2)基因表达的重组干扰载体,为进一步研究人SMS1和SMS2基因与动脉粥样硬化及其他疾病发生的关系提供研究基础。方法根据基因序列数据库中报道的人SMS1和SMS2基因序列及短发夹RNA(shRNA)设计原则,设计并合成用于构建人SMS沉默载体的DNA寡核苷酸,应用基因重组技术分别和线性化pSUPER干扰载体连接,构建了pSUPER-SMS1和pSUPER-SMS2重组干扰载体,并用EcoRI和HindIII限制性内切酶同时处理重组载体pSUPER-SMS1和pSUPER-SMS2,随后测序,以鉴定重组干扰载体含有目的序列。同时为检测重组干扰载体对人SMS1和SMS2基因的干扰效率,本研究利用脂质体(Lipofectamine 2000)分别转染肝癌细胞HepG2,于48h后分别检测HepG2细胞SMS1和SMS2mRNA的转录水平与SMS酶活(薄层层析法,TLC)。结果所筛选到的重组质粒pSUPER-SMS1和pSUPER-SMS2经EcoRⅠ和HindⅢ双酶切后获得了281bp DNA片段,且测序结果和理论设计的DNA寡核苷酸一致;随后检测转染HepG2细胞后SMS1和SMS2的mRNA表达水平。实验结果显示SMS1和SMS2的表达分别下降了45%和67%(P<0.001,n=3),而TLC法的酶活结果检测显示,SMS1和SMS2干扰后的SMS酶活均有显著下降,分别下降了56%和63%(P<0.001,n=3)。结论成功构建了人SMS1以及SMS2基因的沉默载体即pSUPER-SMS1和pSUPER-SMS2。Objective The construct the human sphingomyelin synthase 1 （SMS1） and sphingomyelin synthase 2 （SMS2） interference vector,so as to provid research foundation for further study on relationship between SMS1 and SMS2 gene and atherosclerosis or other related diseases. Methods According to the sequence of SMS1 and SMS2 from the database of gene and rule of designing the shRNA,we designed and synthesized the DNA sequence,then ligated with the linear pSUPER vetor and constructed the silent vector of pSUPER-SMS1 and pSUPER-SMS2.To ensure that the recombinant silent vector included the targed sequence,we double-digested the vecor by the EcoRⅠ and HindⅢ restriction enzyme and sequenced the recombinant vetor by gene company.Meanwhile,to detect interference efficiency of the recombinant silent vector,the recombinant vector was transfected the HepG2 cell by Lipofectamine 2000,respectively,after 48 h,then we investigated the mRNA levels of human SMS1 and SMS2, as well as SMS activity,respectively. Results We acquired the 281 bp DNA fragment after the recombinant vector,pSUPER-SMS1 and pSUPER-SMS2,were double digested by EcoRⅠ and HindⅢ.The sequencing result was the same to the theoretic DNA sequence.The result of transfecting the HepG2 cells showed that it obviously down-regulated the mRNA expression of SMS1 and SMS2,the rate was 45% and 67% respectively （P〈0.001,n=3）.The enzyme activity of SMS also obviously decreased,and the rate was 56% and 63% respectively （P〈0.001,n=3）. Conclusions The study was successed in constructing the human SMS1 and SMS2 interference vector,pSUPERSMS1and pSUPERSMS2.

关 键 词：鞘磷脂合酶 pSUPER干扰质粒 HEPG2细胞 

分 类 号：Q559[生物学—生物化学]