| Abstract: | 本計畫為製造單純皰疹病毒第一型UL49.5基因產物,之後利用製造出之蛋白質製備單株及多株抗體,於完成抗體製備再以此確認單純皰疹病毒第一型UL49.5基因產物之特性,包括分子量、製造時期、於病毒顆粒位置及轉譯後之修飾作用等。目前已依照研究方法之方略二合成,依序為:(1)HSV-1a,a.a. 22-43. (22 amino acids);(2)HSV-1b,a.a. 43-57. (15 amino acids);(3) HSV-1c,a.a. 22-57. (36 amino acids),並將合成之連接KLH (Keyhole limpet hemocyanin),之後以Balb/c mice及New Zealand strain rabbits為動物來源製備抗體。其中單株抗體之製造,第二組及第三組已完成融合瘤之製造,分別獲得39及41個克隆,但第一組之老鼠於免疫過程中全部死亡。而多株抗體之製造,第三組亦已完成兩次抗原之注射,但第一、二組之兔子亦不幸於免疫過程中死亡。後續之工作包括完成單株及多株抗體之製備與UL49.5基因產物之特性分析將持續進行。
The aims of this study are producing poly- and monoclonal antibodies against UL49.5 and characterizing the basic biochemical properties to reconfirm previous findings. According to the hydropathy profiles (Kyte & Doolittle, 1982) of the UL49.5 proteins of HSV-1 (Barnett et al., 1992, Barker & Roizman, 1992), the amino acids fragments chosen to be synthesized are listed below. (1) HSV-1a. a.a. 22-43. (22 amino acids); (2) HSV-1b. a.a. 43-57. (15 amino acids); (3) HSV-1c. a.a. 22-57. (36 amino acids). In this approach, KLH (Keyhole limpet hemocyanin, Pierce) kit was used. Two fusions were made for HSV-1/b and HSV-1/c respectively and 39 and clones were obtained. Theses clones are under screening. However, unfortunately the mice immunized with HSV-1a and the rabbits immunized with HSV-1a and HSV-1b were died during the immunization process. Except for the identification and characterization of the produced antibodies themselves, the characterization analyses for HSV-1 UL49.5 product include trypsin treatment, PAA (phosphonoacetic acid) treatment, glycosylation inhibitor treatment, reducing agents treatment, lectin binding analysis, immunoprecipitation for modification analysis, and neutralization test are to be continued. |
