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標題: 假性狂犬病毒醣蛋白gE的表現及應用
Expression and application of the pseudorabies virus (PRV) glycoprotein E (gE)
作者: 楊于萱
Yang, Yu-Shiuan
關鍵字: PRV;假性狂犬病毒;glycoprotein E;monoclonal antibody;醣蛋白gE;單源抗體
出版社: 獸醫微生物學研究所
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假性狂犬病毒 (pseudorabies virus, PRV) 屬於a- 疱疹病毒亞科
(Alphaherpesvirinae),是造成假性狂犬病的病原。豬是PRV 的自然宿主,感
染PRV 會造成仔豬高致死率,以及懷孕母豬的流產或死產,其盛行率遍及全
式,gE 缺損之疫苗已廣泛使用,其優點為可測定豬隻血清中是否具有抗gE
的抗體,來區分受感染及已免疫之動物,因此gE 具有發展成為快速診斷工具
之潛力。本研究即針對gE 基因進行選殖,將PRV TNL 株gE 基因大小為804
bp 亦即表現N 端268 個胺基酸的基因片段,轉殖於pET28 表現載體中,並於
E. coli 中進行大量表現。所得之gE 重組蛋白 (gEN268) 經由SDS-PAGE 及
Western blot 分析之結果顯示,於45 kDa 處可被抗PRV 豬陽性血清所辨識,
確認其具有正確抗原性。將gEN268 做為抗原並免疫BALB/c 小鼠,利用融合
瘤技術進而製備單源抗體。融合瘤細胞以間接免疫螢光染色法 (indirect
immunofluorescence assay, IFA) 進行篩選,並進一步進行單株化。單株化後之
融合瘤細胞以IFA 進行篩選,並以Western blot 分析單源抗體之特異性,證實
所製備之單源抗體能專一性辨認PRV gE 蛋白。此外,本研究針亦對gE 上游
基因特定片段進行選殖,並分析具啟動子活性之功能區。將gE 基因上游不同
DNA 片段轉殖於CMV 啟動子缺損的重組報告載體 (pEGFPDCMV) 中,分別
構築出pEGFP/gEp166、 pEGFP/gEp425 及 pEGFP/gEp1200 等三個重組質
體,將各重組報告質體分別轉染至PK-15 細胞中進行短暫表現,於轉染後48
小時以倒立螢光顯微鏡觀察細胞是否能表現綠色螢光蛋白 (EGFP) 而呈現綠
色螢光。結果顯示gE 基因上游1 到166 核苷酸序列即具有有效的啟動子功能

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.
其他識別: U0005-0807200818422100
Appears in Collections:微生物暨公共衛生學研究所

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