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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 a recombinant plasmid pTracer CMV2-CRP expressing human C-reactive protein (CRP) and observe its expression in human umbilical vein endothelium (HUVEC) Effects of Lectin-type oxidized LDL receptor-1 (LOX-1) and Tissue factor (TF) on the expression of Lectin-type oxidized low density lipoprotein receptor-1 Methods The CDS sequence of CRP gene was amplified by PCR using the plasmid pCR-BluntⅡ-TOPO-CRP as a template and cloned into pTracer CMV2 vector. The recombinant plasmid pTracer CMV2-CRP was transformed into competent E. coli DH5α and transfected into HUVEC. (Transfected with pTracer CMV2-CRP), negative control group (transfected with pTracer-CMV2) and normal control group. The cells in each group were screened by G418 resistance. The over-expression of CRP gene and the expression of CRP Expression on the transcription and protein level of LOX-1 and TF in HUVECs. Results The recombinant eukaryotic expression plasmid pTracer CMV2-CRP was confirmed by double enzyme digestion and sequencing. In the experimental group, CRP gene was overexpressed and the transcription and protein levels of LOX-1 and TF gene were significantly higher than those in normal control group and negative control group (P <0.05). Conclusion The recombinant eukaryotic expression plasmid pTracerCMV2-CRP of human CRP gene was successfully constructed and CRP was overexpressed in HUVECs. The expression of LOX-1 and TF in HUVECs was significantly increased. To further elucidate the role of CRP in the process of atherosclerosis Function provides a new experimental basis.