摘要
为研究猪传染性胃肠炎病毒(TGEV)N蛋白的表达对宿主细胞功能的影响,本研究通过构建TGEV N基因的重组表达质粒p EGFP-N,将其转染于猪睾丸(ST)细胞中,并在活细胞状态下直接观察GFP-N蛋白在ST细胞中的表达与定位。结果显示,转染后4 h^5 h出现荧光蛋白表达,24 h达到高峰,TGEV的N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TGEV N蛋白主要定位于ST细胞的内质网上面。以G418筛选4周后,转染细胞形成稳定的细胞株,RT-PCR和western blot验证TGEV N蛋白的m RNA及其蛋白在ST细胞中稳定表达,提示本研究建立了稳定表达的TGEV N蛋白的ST细胞株,为进一步研究TGEV N蛋白对ST细胞的生理功能的影响及其在TGEV致病过程中的作用奠定了基础。
In order to study the effects of transmissible gastroenteritis virus（TGEV） N protein on its host cells,the recombinant expression plasmid p EGFP-N was constructed and transfected into the ST cells with LipofectamineTM2000.Then the expression and localization of fusion protein GFP-N were observed by fluorescent microscope.The results showed that TGEV N protein was remarkably expressed in 4 to 5 hrs and achieved the peak at 24 hrs after the transfection,and this protein was localized in the cytoplasm of transfected ST cells by fluorescence microscope observation.Furthermore,the detection of confocal microscopy showed that N protein was mainly expressed in the endoplasmic reticulum.While,the cell lines stably expressed N protein was established from the ST cells transfected with p EGFP-N in present of G418.RT-PCR and western blot results showed that TGEV N protein was expressed in the cell lines,indicating that the cell lines were established successfully,which might lay a foundation for further study of the on TGEV N protein.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2016年第2期101-104,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31302105)
河南科技大学博士生基金(9001732)
关键词
猪传染性胃肠炎病毒
N蛋白
亚细胞定位
细胞株
transmissible gastroenteritis virus
N protein
subcellular location
cell lines
