Expression and application of the pseudorabies virus (PRV) glycoprotein E (gE)

• Main Authors: Yu-Shiuan Yang, 楊于萱
• Other Authors: Chienjin Huang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/74951610358553743391

碩士 === 國立中興大學 === 獸醫微生物學研究所 === 96 === Pseudorabies virus (PRV), an alphaherpesvius, is the causative agent of pseudorabies in swine. The pig is the natural host of PRV, which is characterized by a fatal infection in piglets and abortion in pregnant sows. Pseudorabies is an economically important swine disease worldwide, and vaccination is now used widely in the control of this disease. The gE deleted PRV vaccine strain has been developed, which provides an important advantage to distinguish infected animals from vaccinated ones. Thus, PRV glycoprotein E (gE) has been recognized as a suitable diagnostic reagent for pseudorabies. In order to produce gE protein in a large amount, a 804 bp DNA fragment encoding gE epitopes of PRV TNL strain was cloned in E. coli pET28a expression vector. SDS-PAGE and Western blotting assay showed that the fusion protein (gEN268) was approximately 45 kDa and could be recognized by the swine anti-PRV serum. In addition, the gEN268 protein was used as an antigen to immunize BALB/c mice for developing monoclonal antibody specific to PRV gE by hybridoma techniques. The hybridomas secreting specific antibody against PRV gE were characterized by indirect immunofluorescence assay and Western blotting assay. Furthermore, in order to study the promoter function of gE gene, several defined portions of the upstream of gE gene start codon replaced the CMV promoter of pEGFP-N3 reporter vector and three recombinant plasmids pEGFP/gEp166, pEGFP/gEp425 and pEGFP/gEp1200 were generated. The expression of enhanced green fluorescence protein (EGFP) driven by the gE promoter in transfected PK-15 cells was visualized with a fluorescence microscope at the 48 h post-transfection. The results indicated that the region of nucleotide residues 1-166 upstream of gE start codon possessed sufficient promoter activity for expression of PRV gE.