多房棘球绦虫severin基因过表达慢病毒载体的构建及对人正常肝细胞侵袭、迁移能力的影响

Construction of a lentiviral vector overexpressing the severin gene of Echinococcus multilocularis and its effect on invasion and migration in human liver cells

目的构建多房棘球绦虫肌切蛋白(Em severin)基因过表达慢病毒载体,并分析其对人正常肝细胞L02侵袭、迁移能力的影响。方法扩增Em severin基因片段,并将其插入慢病毒载体pCDH-CMV-MCS-EF1-copGFP,构建重组慢病毒pCDH-Em severin。用pCDH-Em severin感染293T细胞,进行病毒包装并检测病毒滴度。以MOI=50的慢病毒感染人正常肝细胞L02,采用RT-qPCR和Western blot检测Em severin mRNA和蛋白表达水平,并通过Transwell和划痕试验检测过表达Em severin对L02细胞侵袭、迁移能力的影响。结果成功构建重组慢病毒pCDH-Em severin,感染293T细胞并进行包装后,获得滴度为1×10~8 TU/ml的pCDH-Em severin慢病毒。RT-qPCR和Western blot检测pCDH-Em severin感染组Em severin mRNA和蛋白表达水平显著高于空白组和空载病毒组(F值分别为98.731和86.074,P<0.05)。Transwell和划痕试验结果表明,过表达Em severin可增强人正常肝细胞L02的侵袭、迁移能力(F值分别为15.439和53.082,P<0.05)。结论成功构建重组慢病毒pCDH-Em severin,过表达Em severin可增强人正常肝细胞L02的侵袭、迁移能力,可为研究Em severin在泡球蚴的侵袭。

Objective To construct a lentiviral vector overexpressing the severin gene of Echinococcus multilocularis(E.multilocularis severin) and to observe its effect on invasion and migration in L02 human liver cells.Methods The severin gene of E.multilocularis was amplified and inserted into the lentiviral vector pCDH-CMV-MCS-EF1-copGFP to construct the recombinant lentivirus pCDH-Em severin,which was transfected into 293 T cells for lentivirus packaging and determination of the viral titer.The recombinant lentivirus pCDH-Em severin was transfected into L02 human liver cells with a multiplicity of infection of 50.Levels of expression of mRNA and protein of E.multilocularis severin were detected using RT-qPCR and Western blotting,respectively.The effects of Em severin on invasion and migration in L02 human liver cells were identified using a Transwell assay and cell scratch test.Results The recombinant lentivirus pCDH-Em severin was successfully constructed,and 293 T cells were infected and packaged to obtain pCDH-Em severin lentivirus,with a titer of 1×10~8 TU/ml.The results of RT-qPCR and Western blotting indicated that the levels of expression of mRNA and protein of E.multilocularis severin in a group infected with the pCDH-Em severin lentivirus were significantly higher than those in the control group and the group infected with an empty lentivirus vector(F: 98.731 and 86.074,respectively,P<0.05).More importantly,results of a Transwell assay and cell scratch test indicated that overexpression of E.multilocularis severin increases invasion and migration in L02 human liver cells(F: 15.439 and 53.082,respectively,P<0.05).Conclusion The recombinant lentivirus pCDH-Em severin was successfully constructed,and overexpression of E.multilocularis severin increased invasion and migration in L02 human liver cells.These findings have laid the foundation for further study of the roles and mechanisms of severin in the process of invasion and migration by E.multilocularis.