摘要
目的构建血红素加氧酶1基因(HO-1)真核双表达载体pCDNA3.1-ho-1:t2a:egfp,并在小鼠肾足细胞MPC5中过表达,研究HO-1在脂代谢紊乱模型下的抗炎作用。方法取生长良好的人胚胎肾细胞HEK293t,提取总RNA反转录得到cDNA文库,扩增得到HO-1基因目的片段。将目的片段和载体进行双酶切,超滤管回收后T4-ligase连接,得到真核表达质粒命名为pCDNA3.1-ho-1:t2a:egfp。将载体进行菌液PCR、双酶切和测序鉴定后,转染小鼠肾足细胞MPC5,通过荧光显微镜和Western blot检测HO-1以及炎性因子表达情况。结果以HEK293t为来源扩增得到的目的片段凝胶电泳位置正确;筛选得到的载体菌液PCR、双酶切凝胶电泳以及测序结果正确;转染细胞后,荧光显微镜和Western blot结果显示质粒转染成功,并且减少了炎性因子IL-18、IL-1β表达。结论成功构建pCDNA3.1-ho-1:t2a:egfp双表达质粒,为糖尿病脂代谢紊乱细胞模型后续相关实验奠定基础。
Objective To construct the eukaryotic double expression vector pCDNA3.1-ho-1:t2a:egfp of heme oxidase 1 gene(HO-1)and overexpress it in mouse glomerular podocytes MPC5 to study the anti-inflammatory effect of HO-1 in the model of lipid metabolism disorder.Methods The total RNA from human embryonic kidney HEK293t cells with good growth was extracted and inversely transcribed into cDNA,which was subsequently amplified to obtained the target fragment of HO-1 gene.The target fragment and vector were digested by double enzyme,and then ligated by T4-ligase after their recovery with ultrafiltration tube.The eukaryotic expression plasmid was named as pCDNA3.1-ho-1:t2a:egfp.The vector was identified by PCR,double enzyme digestion and sequencing,and then transfected into mouse glomerular podocytes MPC5.The expressions of HO-1 and inflammatory factors were detected by fluorescence microscopy and western blot.Results The target fragments amplified form HEK293t showed the correct size and position in the gel electrophoresis,and were futher confirmed by the results of the selected bacterial liquid PCR,double enzyme digestion gel electrophoresis and sequencing.After transfection,the results of fluorescence microscopy and western blot showed that the plasmid was successfully transfected,and the expressions of inflammatory factors IL-18 and IL-1β were reduced.Conclusion The double expression plasmid of pCDNA3.1-ho-1:t2a:egfp was successfully constructed,and laid the foundation for the subsequent experiments of diabetic lipid metabolism disorder cell model.
作者
唐明秋
宋熠
倪萍
赵宝清
TANG Ming-qiu;SONG Yi;ZHAO Bao-qing(School of Pharmacy,Xianning Medical College,Hubei University of Science and Technology,Xianning Hubei 437100,China)
出处
《湖北科技学院学报（医学版）》
2023年第2期110-113,119,共5页
Journal of Hubei University of Science and Technology(Medical Sciences)
基金
湖北省教育厅中青年人才项目(Q20202803)
糖尿病心脑血管病变湖北省重点实验室糖尿病专项基金(2020TNB10,2022TNB11)
湖北科技学院博士启动基金项目(BK202010)。
关键词
血红素加氧酶1基因
载体构建
真核表达
抗炎作用
Heme oxygenase 1 gene
Vector construction
Eukaryotic vector expression
Anti-inflammatory
