Huang C, Lin Y S, Cheng J W, Chang T J
Graduate Institute of Veterinary Microbiology, National Chung Hsing University, Taichung, Taiwan, ROC.
J Virol Methods. 1997 Jul;66(2):219-26. doi: 10.1016/s0166-0934(97)00069-4.
The immediate-early (1E) gene of pseudorabies virus (PRV) expresses immediately upon infection, a phosphorylated protein (immediate-early protein, IE180) that can transactivate viral other genes and plays an essential role in regulating viral gene expression. In order to detect and localize IE180 in infected cells early on, this gene was cloned for overexpression, and the expressed products were applied to generate specific antibodies against IE180 protein. Two recombinant expression plasmids pN and pNB were constructed by cloning the IE gene onto pET 30a(+) expression vector via NcoI and BamHI sites. Plasmid pN contains the 1.8-kb NcoI-NcoI fragment of IE gene coding for the N-terminus of 616 amino acid residues, while pNB contains the 2.8-kb NcoI-Bam HI fragment coding for the rest of the IE180 protein. Both pN and pNB were transformed, respectively, into E. coli cells and produced large amounts of IE protein products during induction with 1 mM IPTG. The expressed IE proteins for pN and pNB were 60 kDa and 100 kDa in size, respectively. These expression products were purified and then used as antigens to immunize mice for preparing specific antibodies against PRV IE180 protein. The specificities of the mice immune sera were confirmed by their abilities to react with IE180 protein present in the PRV infected cells in the Western immunoblotting assay. Furthermore, immunoperoxidase staining of PRV infected cells undertaken with these antisera revealed the subcellular distribution of the IE proteins in the infected cells and also demonstrated their transportation from the cytoplasm to the nucleus during infection.
伪狂犬病病毒（PRV）的立即早期（IE）基因在感染后立即表达一种磷酸化蛋白（立即早期蛋白，IE180），该蛋白可反式激活病毒的其他基因，并在调节病毒基因表达中起重要作用。为了早期在感染细胞中检测和定位IE180，克隆该基因进行过表达，并将表达产物用于制备针对IE180蛋白的特异性抗体。通过将IE基因经NcoI和BamHI位点克隆到pET 30a(+)表达载体上，构建了两个重组表达质粒pN和pNB。质粒pN包含IE基因1.8 kb的NcoI - NcoI片段，编码616个氨基酸残基的N端，而pNB包含2.8 kb的NcoI - BamHI片段，编码IE180蛋白的其余部分。pN和pNB分别转化到大肠杆菌细胞中，并在1 mM IPTG诱导下大量产生IE蛋白产物。pN和pNB表达的IE蛋白大小分别为60 kDa和100 kDa。这些表达产物经纯化后用作抗原免疫小鼠，以制备针对PRV IE180蛋白的特异性抗体。在蛋白质免疫印迹分析中，小鼠免疫血清与PRV感染细胞中存在的IE180蛋白发生反应的能力证实了其特异性。此外，用这些抗血清对PRV感染细胞进行免疫过氧化物酶染色，揭示了感染细胞中IE蛋白的亚细胞分布，并证明了它们在感染过程中从细胞质转运到细胞核。
