摘要
病毒蛋白VP2是兔出血症病毒(Rabbit Hemorrhagic Disease Virus,RHDV)的一种次要结构蛋白。本文旨在构建VP2与绿色荧光蛋白融合的真核表达载体,进而利用它研究VP2在细胞内的定位情况。首先在大肠杆菌中表达GST-VP2融合蛋白,然后用纯化的重组蛋白免疫小鼠制备抗VP2的抗血清。对制备的抗VP2抗血清进行特异性分析,再利用该抗体对转染pEGFP-VP2的BHK-21细胞进行检测,以研究VP2蛋白在细胞内的定位情况。结果表明,pEGFP-VP2转染BHK-21细胞后,不仅VP2蛋白得到了成功表达,而且通过检测偶联的绿色荧光蛋白可判断出VP2蛋白主要分布于BHK-21细胞的细胞质。本研究成功构建了VP2与绿色荧光蛋白融合的表达载体,并在真核细胞内实现了表达,通过检测荧光蛋白,发现VP2主要在细胞质内表达并分布,为深入探讨VP2的生物学功能奠定了基础。
Virus protein VP2 is a minor structural protein of rabbit hemorrhagic disease virus （RHDV）. The objective of the present study was to study the expression and intracellular localization of VP2 in eukaryotic cells by fusion with enhanced green fluorescent protein （EGFP）. The GST-VP2 fusion protein expressed in E. coli was purified and inoculated and the specificity to VP2 was determined. into mice. The polyclonal murine antibody was prepared To explore the intracellular localization of VP2, the recombinant plasmid （pEGFP-VP2）fused with EGFP was constructed and transfected into BHK-21 cells. The expression of EGFP-VP2 protein was detected by Western blot assay. The intracellular localization of VP2 was observed under fluorescence microscope. Besides, the localization of VP2 was further examined by staining cells with DAPI. The recombinant plasmids were constructed and the fusion proteins were expressed in BHK-21 cells. The VP2 and EGFP were co-localized in BHK-21cells and VP2 was primarily localized in cytoplasm as determined by using indirect immunofluorescence assay （IFA） and DAPI staining. The construct of fusion expression vector of pEGFP-VP2 and expression in eukaryotic cells have laid the foundation for studying the biological function of VP2.
出处
《中国动物传染病学报》
CAS
2009年第2期1-7,共7页
Chinese Journal of Animal Infectious Diseases
基金
国家自然科学基金项目(308000453067007430870114)
家畜疫病病原生物学国家重点实验室开放基金资助
关键词
兔出血症病毒
VP2
次要结构蛋白
细胞内定位
Rabbit hemorrhagic disease virus（RHDV）
VP2
minor structural protein
intracellular localization
