稳定表达猪瘟病毒NS5A蛋白的细胞系的建立

Establishment of the cell lines for stable-expression of NS5 Aprotein of classical swine fever virus

为了探索猪瘟病毒非结构蛋白5A(NS5A)的功能,成功扩增了猪瘟病毒NS5A基因的DNA片段并构建了猪瘟病毒NS5A基因的重组表达质粒pGFP-NS5A和pNS5A-GFP。然后,将阳性质粒pGFPNS5A、pNS5A-GFP和对照质粒pEGFP-C1在Lipofectamine 2000介导下转染猪血管内皮细胞(SUVEC),活细胞状态下直接观察GFP-NS5A和NS5A-GFP蛋白在SUVEC细胞中的表达与定位。结果显示,猪瘟病毒NS5A蛋白主要定位于细胞质中;进一步的激光共聚焦共定位显示,NS5A蛋白主要定位于SUVEC细胞的内质网上面。在此基础之上,通过含有1 500μg/mL新霉素(G418)的抗性培养基筛选出稳定表达猪瘟病毒NS5A蛋白的细胞克隆。RT-PCR和Western-blot检测结果表明,猪瘟病毒NS5A蛋白可以在SUVEC细胞中稳定转录和表达。上述结果表明,本研究建立了稳定表达猪瘟病毒NS5A蛋白的SUVEC细胞株,为进一步研究NS5A蛋白对SUVEC细胞的生理功能的影响及其在猪瘟病毒致病过程中的作用奠定了基础。

In order to probe the unknown function of nonstructural protein 5Aof classical swine fever virus(CSFV),the NS5 Agene was successfully amplified from the total RNAs of CSFV infected cells by reverse transcription polymerase chain reaction(RT-PCR)and cloned into the pEGFP-C1 and pEGFP-N1 expression vectors according the vector information,respectively.The recombinant plasmids pGFP-NS5 A,pNS5A-GFP and pEGFP-C1 were transfected into SUVEC by Lipofectamine 2000,respectively.In result,CSFV NS5 Aprotein localized in the cytoplasm.Further study showed that CSFV NS5 Aprotein located in the endoplasmic reticulum by confocal laser scanning microscope.Then the stably transfected cell lines expressing fusion proteins GFP-NS5 A,NS5A-GFP and GFP were screening in the selective media containing1 500μg/mL.RT-PCR and Western-blot analysis showed that CSFV NS5 A gene was successfully expressed in SUVEC.In conclusion,the result of this study lay the foundation for study on the effect of NS5 Aprotein in the pathogenesis and mechanism of CSFV.