牛病毒性腹泻病毒E2蛋白的亚细胞定位研究

Subcellular Localization Analysis of Bovine Viral Diarrhea Virus E_2 Protein

为深入研究牛病毒性腹泻病毒E_2蛋白的生物学功能,了解其在哺乳动物细胞中的亚细胞定位情况,采用RT-PCR扩增牛病毒性腹泻病毒Changchun184株E_2基因,将其克隆至真核表达载体pc DNA3.1/V5His A中,并进行酶切鉴定和测序分析,将构建的重组质粒pc DNA3.1/V5His A-E_2经脂质体介导转染至MDBK细胞。继续培养48h后,在激光共聚焦显微镜下观察E_2蛋白在细胞中的亚细胞定位情况。结果表明,E_2蛋白在细胞核与细胞质中均有表达,且绿色荧光呈点状均匀分布。该研究为进一步研究牛病毒性腹泻病毒E_2蛋白的相关功能奠定了基础。

This experiment was designed to make a thorough study on the biological function of bovine viral diarrhea virus E_2 protein by investigating its subcellular localization in mammalian cells. The E_2 gene fragments of Changchun184 were amplified by RT- PCR,cloned into eukaryotic expression vector pc DNA3. 1 / V5 His A,and then identified by restriction enzymes digestion and sequencing analysis. The subcellular location of E_2 protein in MDBK cells was observed by confocal laser scanning microscope after that the recombinant plasmid pc DNA3. 1 / V5 His A- E_2 was transfected into MDBK cells by lipofectin for 48 h. The results showed that the E_2 protein was expressed in both the nucleus and cytoplasm,and most of the green fluorescence was distributed in a speckled pattern. All of these results will be as a basis for the further research on E_2 protein＇s functions.