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 |
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引用本文: | 冯艳,王月玲,李明远,江滟,李婉宜,杨远,王保宁,冯伟,张强,蒋忠华.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[J].西部医学,2009,21(7):1072-1075. |
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作者姓名: | 冯艳 王月玲 李明远 江滟 李婉宜 杨远 王保宁 冯伟 张强 蒋忠华 |
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作者单位: | 1. 四川大学华西基础医学与法医学院微生物学教研室,四川,成都,610041 2. 四川大学华西基础医学与法医学院微生物学教研室,四川,成都,610041;贵阳医学院微生物学教研室,贵州,贵阳,550004 |
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摘 要: | 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Xho工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mBDl-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-定的抗流感病毒作用。为进-步深入研究mBDI-mBD3的生物学特性及其抗流感病毒作用机制奠定了基础。
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关 键 词: | mBDI—mBD3 RT—PCR 真核表达 免疫荧光 重叠-PCR TCID50 |
Construction of eukaryotic expression vector with mBD1-mBD3 genes and exploring its activity against influenza virus in vitro |
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Institution: | FENG Yan , WANG Yue-ling , Li Ming-yuan ,et al (Department of Microbiology, West China of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China; 2. Department of Microbiology, Guiyang Medical college, Guiyang 550004, China) |
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Abstract: | 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. |
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Keywords: | mBD1-mBD3 RT-PCR TCID50 |
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