摘要
目的构建小鼠C-反应蛋白基因重组质粒pIRES-CRP,观测其对血管平滑肌细胞TNF-α表达的影响。方法用RT-PCR法,以小鼠肝组织RNA为模板,扩增获得编码C-反应蛋白(C-reactive protein,CRP)的全长基因。将CRP基因克隆入真核表达质粒pIRES中,构建重组真核表达质粒pIRES-CRP。将重组质粒转染HeLa细胞,通过RT-PCR法检测CRPmRNA的表达,Western blot检测CRP蛋白的表达。将阳性质粒转染小鼠血管平滑肌细胞,通过Western blot检测CRP对TNF-α表达的影响。结果经酶切鉴定、PCR和测序分析结果表明,所克隆的CRP基因与报道结果完全一致,重组体的连接方向正确,阅读框与预期完全一致,真核表达质粒pIRES-CRP构建成功。将重组质粒pIRES-CRP瞬时转染Hela细胞,通过RT-PCR和Western blot证实CRP基因得到表达。将阳性质粒转染小鼠血管平滑肌细胞,TNF-α的表达明显高于空白对照组和空载体组(P<0.01)。结论成功构建小鼠CRP基因重组质粒pIRES-CRP,CRP基因能在人子宫颈癌Hela细胞中正常转录和翻译,表达的蛋白能与CRP的特异抗体反应。CRP能明显上调小鼠血管平滑肌细胞TNF-α的表达,这为解释CRP在冠状动脉的形成过程中起着促炎作用提供了新的实验依据。
Objective To obtain the full length gene of C-reactive protein（CRP） from mouse liver cells,to construct the recombination plasmid pIRES-CRP and to investigate its effect on TNF-α Expression in mouse aortic smooth muscle cells.Methods CRP genes were obtained by RT-PCR with total RNA of mouse as the template and cloned into the eukaryotic expression plasmid pIRES to construct the recombinant plasmid pIRES-CRP.After the recombinant plasmid pIRES-CRP was transfected into Hela cells,the expression of CRP genes was detected by using RT-PCR and Western blot.The expression of TNF-α genes was detected by using Western blot when the recombinant plasmid pIRES-CRP was transfected into mouse aortic smooth muscle cells.Results Restriction enzyme analysis,PCR and sequencing results showed that the recombinant plasmid pIRES-CRP was constructed successfully.After the recombinant plasmid pIRES-CRP was transfected into Hela cells,the expression of CRP genes could be detected by RT-PCR and Western blot.The expression of TNF-α genes was significantly higher than that in the control group and the vector group（P0.01） when the recombinant plasmid pIRES-CRP was transfected into mouse aortic smooth muscle cells.Conclusion The eukaryotic expression plasmid pIRES-CRP was constructed successfully and CRP genes of mice were expressed in Hela cells by this vector.The expression of TNF-α genes of mouse aortic smooth muscle cells can be promoted by CRP,which provides new evidence for the pro-inflammatory action of CRP in atherogenesis.
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2011年第1期11-16,共6页
Chinese Journal of Histochemistry and Cytochemistry
