mBD1-mBD3融合基因真核表达载体构建及体外抗流感病毒初步研究

Construction of eukaryotic expression vector with mBD1-mBD3 genes and exploring its activity against influenza virus in vitro

目的构建小鼠β-防御素1和β-防御素3(mBD1,mBD3)融合基因的真核表达载体,研究mBD1-mBD3融合蛋白的抗流感病毒作用。方法通过RT-PCR和重建PCR方法构建mBD1-mBD3融合基因;经EcoRⅠ和XhoⅠ双酶切后插入相同酶切的pcDNA3.1(+),构建成重组质粒pcDNA3.1(+)/mBD1-mBD3,对重组质粒进行PCR、酶切和测序鉴定;将构建好的真核表达载体转染MDCK细胞,G418筛选稳定表达株,RT-PCR和免疫荧光染色法鉴定胞内mBD1-mBD3的表达。用流感病毒感染稳定表达细胞株,TCID50测定并分析抗流感病毒作用。结果成功克隆到mBD1-mBD3基因,并构建了真核表达载体pcDNA3.1(+)/mBD1-mBD3。RT-PCR和间接免疫荧光证实重组质粒可以在细胞内表达。实验组的TCID50显著高于对照组,初步显示出mBD1-mBD3的抗流感病毒作用。结论本研究成功构建了pcDNA3.1(+)/mBD1-mBD3真核表达载体,且能在MDCK细胞中稳定表达,表达产物具有一定的抗流感病毒作用。为进一步深入研究mBD1-mBD3的生物学特性及其抗流感病毒作用机制奠定了基础。

AIM To clone murine beta defensin-1 （mBD1） and murine beta defensin-3 （mBD3） genes, construct the eukaryotie expression vectors pcDNA3.1（＋） /mBDI-mBD3, and observe mBDI-mBD3 expression pattern in cells as well as its activity against influenza virus. Methods mI3DI-mBD3 fusion genes were amplified by RT-PCR and Over-lap PCR, then fusion fragment was inserted into eukaryotic expressing vector pcDNA3.1（＋） The recombinant plasmid pcD- NA3. 1（＋）/mBDI-mBD3 was identified and transfeeted into MDCK cells. The mBDI-mBD3 steady expression pattern was confirmed by RT-PCR and immunofluorescence assay. The activity against influenza virus with the plasmid was also determined by TCID50. Results About 350bp product of mBD1-mBD3 was amplified by overlap-PCR. The eukaryotic expression vector, called as peDNA3. 1（＋）/mBD1-mBD3, was constructed corectly. Stable MDCK cells transfected by peDNA3. 1（＋）/mBD1-mBD3 were got by G418 screening. The fusion protein of mBD1-mBD3 was detected in the MDCK cells. The TCID50 of experimental group was lower than that of control group obviously. Conclusion The construction of the eukaryotic vectors of the peDNA3. 1 （＋）/mBD1-mBD3 was completely successful. The pcDNA3. 1 （＋）/ mBDI-mBD3 could expressed mBDI-mBD3 protein in MDCK cells stably. The mBD1-mBD3 had the activity against influenza virus effectually. These results established a solid foundation for further studying on the biological properties and anti-virus mechanism of the mBD1-mBD3 fusion protein.