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Characterization of Monoclonal Antibody against the pseudorabies virus UL54 Protein
|關鍵字:||pseudorabies virus;假性狂犬病毒;UL54;monoclonal antibody;nucleocytoplasmic shuttling;UL54;單株抗體;核質穿梭||出版社:||獸醫微生物學研究所||引用:||吳金英。2003。假性狂犬病毒早期調節基因UL54之選殖與表現及功能分析。國立中興大學獸醫微生物學研究所碩士論文。 陳麗芬。2006。假性狂犬病毒早期蛋白UL54之核停留及輸出訊號的定位。國立中興大學生命科學系學士論文。 黃雅如。2004。假性狂犬病毒早期蛋白UL54 之功能區分析。國立中興大學獸醫微生物學研究所碩士論文。 Ackermann, M., D. K. Braun, L. Pereira, and B. Roizman. 1984. Characterization of herpes simplex virus type 1 a proteins 0, 4, and 27 with monoclonal antibodies. J. Virol. 52:108–118. Alber G., U. M. Kent, and H. Metzger. 1992. Functional comparison of Fc epsilon RI, Fc gamma RII, and Fc gamma RIII in mast cells. J. Immunol. 149:2428–36. Baumeister, J., B. G. Klupp, and T. C. Mettenleiter. 1995. Pseudorabies virus and equine herpesvirus 1 share a nonessential gene which is absent in other herpesviruses and located adjacent to a highly conserved gene cluster. J. Virol. 69:5560–5567. Bogerd, H., R. Fridell, R. Benson, J. Hua, and B. Cullen. 1996. 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Selective inhibition of rat liver nuclear RNA polymerase II by actinomycin D in vivo. Carcinogenesis 1:577-581.||摘要:||
假性狂犬病毒 (pseudorabies virus, PRV) 是一種引起猪隻重要傳染病的alphaherpesvirus，除了高等靈長類以外大部份的哺乳類動物皆可被感染。PRV早期蛋白UL54是一種核質穿梭蛋白由363個胺基酸所組成且分子量約40 kDa，其蛋白序列上帶有核輸入 (nuclear localization signal, NLS) 以及核輸出訊號 (nuclear export signal, NES) 的功能區。本研究之目的為製備特異性抗PRV UL54單株抗體並進一步利用此單株抗體以監測UL54蛋白在PRV感染期間於感染細胞內分佈與表現的情形。首先利用E. coli來大量表現UL54 重組蛋白並經純化後做為免疫BLAB/c小鼠之抗原以進行融合瘤細胞實驗。本實驗成功製備出一株可特異性辨認PRV UL54 蛋白之單株抗體 (8-8G)，進一步針對一系列UL54缺損重組蛋白進行epitope mapping後定出其所辨識抗原決定位 (epitope) 的相關位置乃位於UL54蛋白N端第84到141個胺基酸的區域。此外，利用單株抗體8-8G針對PRV感染的細胞進行間接免疫螢光法 (indirect immunofluorescence assay, IFA) 之結果顯示，隨著感染時間的增加UL54蛋白在細胞質表現後逐漸往細胞核移動並大量累積在細胞核內。但添加了轉錄抑制劑actinomycin D後因使UL54蛋白的核輸入功能受阻導致UL54蛋白逐漸由細胞核移動到細胞質，進一步證實PRV UL54蛋白具有核質穿梭功能。
Pseudorabies virus (PRV) is a porcine alphaherpesvirus that can infect most mammals except for higher-order primates. The PRV UL54 gene encodes a 40 kD protein of 363 amino acids. The early protein UL54 is a nucleo-cytoplasmic shuttling protein with nuclear localization signal (NLS) and nuclear export signal (NES). The purpose of this study was to prepare and characterize the monoclonal antibody against PRV UL54 protein and then to examine the UL54 expression and distribution during PRV infection. The UL54 protein was expressed in E. coli in a large amount and used
as an antigen to immunize BALB/c mice for further hybridoma experiment. One monoclonal antibody (8-8G) was obtained which could specifically recognize the PRV UL54 protein and the epitope recognized by this monoclonal antibody was mapped to the N-terminal amino acid residues 84-141. Furthermore, the transcriptional inhibitor actinomycin D was treated to monitor the intracellular distribution of UL54 protein during PRV infection by indirect immunofluorescence assay (IFA). As infection progressed, UL54 protein localized from the cytoplasm to the nucleus and accumulated abundantly in the nucleus. After the addition of actinomycin D, the UL54 protein moved to the cytoplasm and finally demonstrated more diffuse staining throughout the nucleus and cytoplasm, confirming its shuttling activity.
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