人β-COP-shRNA慢病毒载体构建及干扰效应检测  

Construction of shRNA lentiviral vectors targeting human β-COP and detection of their interference effect

作　　者：马卫列[1] 丁航[1] 李观强[2] 肖娟[1] 张志珍[1] 

机构地区：[1]广东医学院生物化学与分子生物学教研室,东莞医学硕士523808 [2]深圳市龙岗区人民医院检验科,深圳518172

出　　处：《医学研究生学报》2015年第2期136-140,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81170267);广东省自然科学基金(S2011010002984);东莞市高等院校科研机构科技计划项目(2012108102037)

摘　　要：目的采用常规转染试剂难以将人β-COP-shRNA转染至THP-1细胞。构建人β-COP特异性的shRNA慢病毒载体,并测定其对靶基因β-COP的抑制效应。方法设计合成针对人β-COP基因靶点特异的4对shRNA寡聚单链DNA,插入p GMLV-SC1载体中,构建慢病毒重组载体。将重组载体和包装质粒共转染HEK 293T细胞,包装产生慢病毒并测定滴度。用慢病毒感染THP-1细胞,定量PCR和Western blot检测干扰载体对β-COP基因的干扰效果。结果测序证实,β-COP基因的shRNA寡聚核苷酸序列已插入慢病毒载体,转染HEK 293T细胞,经包装得到4种慢病毒,病毒滴度为1×109TU/m L。定量PCR分析显示,4种β-COP-shRNA慢病毒感染的THP-1细胞中β-COP基因mRNA表达量分别为0.831±0.065、0.675±0.079、0.260±0.050、0.497±0.067,较对照(1.000±0.000)明显升高(P<0.01);其抑制率分别为16.9%、32.5%、74.0%和50.3%。Western blot结果表明,4种干扰载体均可抑制β-COP蛋白表达,其中β-COP-shRNA 3的沉默效率为76.4%。结论成功构建了人β-COP-shRNA干扰载体,证实了干扰载体对靶基因有较好的沉默效果。Objective The purpose of this study was to construct a short hairpin RNA（ shRNA） interference lentiviral vector targeting the human β-COP gene and to evaluate its inhibitory effect on β-COP in THP-1 cells. Methods We designed and synthesized4 human β-COP-specific oligonucleotide sequences and inserted them into the p GMLV-SC1 vector to construct a recombinant vector followed by transfection of HEK 293 T cells with the recombinant vector and Lenti-HG Mix to produce lentiviruses and detect the viral content. After infecting the THP-1 cells with the packaged lentiviruses,we analyzed the inhibitory effect of β-COP-shRNA on the β-COP gene by quantitative PCR and Western blot. Results Sequencing confirmed that the β-COP-specific oligonucleotide sequences were inserted into the lentiviral vector and the lentiviruses were packaged in the transfected HEK 293 T cells,with the final viral content of 1 ×109TU/m L. Quantitative PCR showed that the 4 β-COP-shRNA vectors significantly decreased the mRNA expression of β-COP（ P〈0. 01）,with interference rates of 16. 9%,32. 5%,74. 0%,and 50. 3%,respectively. Western blot also indicated their inhibitory effect on the protein expression of β-COP,with an inhibition rate of 76. 4% on β-COP-shRNA3. Conclusion Lentiviral shRNA interference vectors targeting human β-COP were constructed successfully,which could suppress the expression of the human β-COP gene.

关 键 词：人β-COP shRNA干扰载体 脂质代谢 

分 类 号：Q78[生物学—分子生物学]