Cloning and Expression of the Pseudorabies Virus (TNL Strain) Immediate-early Gene

• Main Authors: Lin, Ying-shiow, 林盈秀
• Other Authors: Huang Chien-jin
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/20563332330619904883

碩士 === 國立中興大學 === 獸醫微生物學研究所 === 84 === The immediate-early ( IE ) gene of pseudorabies virus expresses immediately upon infection and does not require prior viral proteins synthesis for its expression. The product of IE gene can transactivate viral early and late genes as well as other cellular genes. Thus, the PRV IE gene plays an important role in initiation of specific gene expressions. The purpose of this study is to characterize the PRV native strain ( TNL ) IE gene transcript and then to construct useful recombinant expression vectors for producing a large amount of IE protein. The virus was first infected to MDBK cells in the presence of 50 ug/ml cycloheximide and then to extract the total mRNA from infected cells. The result of northern blot hybridization using the 8th BamHI fragment of PRV genomic DNA as probe was shown that there was only one IE mRNA produced about 5.6 kb in size. Furthermore, in order to study the structure and functions of IE protein, the IE gene was cloned onto pET 30a(+) expression vector via NcoI and BamHI cloning sites. Two recombinant expression plasmids were obtained in which pN contains IE gene NcoI/NcoI fragment after the initiation codon while pNB contains the rest of the gene ( NcoI/BamHI fragment ). Both pN and pNB were respectively transformed into E. coli and could successfully produce large amounts of IE products in cells during the induction with 1mM IPTG. The expressed IE proteins for pN and pNB were 60 kDa and 100kDa in size respectively. These expression products were further purified by His-bind affinity chromatograhpy and then used as antigens to immunize mice for preparation of specific antisera. The specificity of mice immune sera to IE protein was confirmed by their abilities to react with IE 180 protein in the PRV-infected cells in the western blot analysis. Finally, the immunoperoxidase staining experiment performed with these mouse antisera could localize the IE protein in the PRV-infected cells and also demonstrated its transportation from cytoplasm to nucleus during the infection period.