人C-反应蛋白重组真核表达质粒的构建及其对人脐静脉内皮细胞LOX-1和TF表达的影响

Construction of recombinant eukaryotic expression vector for human C-reactive protein and its effect on expressions of LOX-1 and TF in human umbilical vein endothelial cells

目的构建人C-反应蛋白(C-reactive protein,CRP)重组表达质粒pTracer CMV2-CRP,并观察其在人脐静脉内皮细胞(Human umbilical vein endothelical cells,HUVEC)中的表达及其对凝集素样氧化型低密度脂蛋白受体-1(Lectin-type oxidizedLDL receptor-1,LOX-1)、组织因子(Tissue factor,TF)表达的影响。方法以质粒pCR-BluntⅡ-TOPO-CRP为模板,PCR扩增CRP基因CDS序列,克隆至pTracer CMV2载体中,转化感受态大肠杆菌DH5α,构建重组表达质粒pTracer CMV2-CRP,转染HUVEC,设实验组(转染pTracer CMV2-CRP)、阴性对照组(转染pTracer-CMV2)及正常对照组,各组细胞经G418抗性筛选,RT-PCR及Western b1ot检测CRP基因的过表达效应及CRP的表达对HUVEC中LOX-1和TF转录及蛋白水平的影响。结果重组真核表达质粒pTracer CMV2-CRP经双酶切鉴定及测序证明构建正确。实验组细胞中,CRP基因过表达,且LOX-1和TF基因的转录及蛋白水平明显高于正常对照组和阴性对照组(P<0.05)。结论已成功构建人CRP基因重组真核表达质粒pTracerCMV2-CRP,并在HUVEC中过表达CRP,且明显上调HUVEC中LOX-1和TF的表达,为进一步阐述CRP在动脉粥样硬化形成过程中的作用提供了新的实验依据。

Objective To construct the recombination eukaryotic expression vector pTracer CMV2-CRP for C-reactive protein（CRP） and observe its effect on expressions of lectin-type oxidized low-density lipoprotein receptor-1（LOX-1） and tissue factor（TF） in human umbilical vein endothelial cells（HUVECs）.Methods The CDS sequence of CRP gene was amplified by PCR using plasmid pCR-BluntⅡ-TOP O-CRP as a template and cloned into vector pTracer CMV2.The constructed recombinant plasmid pTracer CMV2-CRP was transfected to HUVECs,using the HUVECs transfected with pTracer-CMV2 as negative control and those untransfected as normal control.The stably transfected HUVECs in various groups were screened with G418,in which the overexpression of CRP gene as well as the effect of CRP expression on LOX-1 and TF mRNA and protein levels in HUVECs were determined by RT-PCR and Western blot.Results Restriction and sequencing proved that recombinant plasmid pTracer CMV2-CRP was constructed correctly.CRP gene was over-expressed in HUVECs transfected with the plasmid,while the LOX-1 and TF mRNA and protein levels were significantly higher than those in normal and negative control groups.Conclusion Eukaryotic expression vector pTracer CMV2-CRP was constructed successfully,and CRP gene was over-expressed in HUVECs,while the expressions of LOX-1 and TF were up-regulated significantly,which provided a new evidence for further study on the role of CRP in atherogenesis.