Title

犬小病毒VP2蛋白和犬瘟熱病毒H蛋白之單株抗體製備及特性分析

Translated Titles

Production and analysis of monoclonal antibodies against the VP2 protein of canine parvovirus and the H protein of canine distemper virus

Authors

鄭年倫

Key Words

犬小病毒 ; 犬瘟熱病毒 ; 單株抗體 ; Canine parvovirus ; canine distemper virus ; monoclonal antibodies

PublicationName

中興大學分子生物學研究所學位論文

Volume or Term/Year and Month of Publication

2012年

Academic Degree Category

碩士

Advisor

劉宏仁

Content Language

繁體中文

Chinese Abstract

犬小病毒 (canine parvovirus, CPV)和犬瘟熱病毒 (canine distemper virus, CDV)都是犬隻重要之傳染性疾病。CPV的結構蛋白鞘是由 VP1、VP2及 VP3所組成,VP2為其主要蛋白,並與宿主的免疫反應有關。CDV主要由六個蛋白構成,病毒之 H蛋白可誘發中和抗體。本研究以原核表現系統分別進行 CPV VP2、CDV H-N端及 CDV H-C端蛋白之表現,所表現之 CPV VP2融合蛋白其分子量約83 kDa,而 CDV H-N端及 CDV H-C端融合蛋白其分子量分別約43 kDa和55 kDa。以 CPV VP2、CDV H-N端及 CDV H-C端之融合蛋白免疫 BALB/c小鼠製備融合瘤細胞,經免疫呈色分析 (enzyme-linked immunosorbent assay;ELISA)初步篩選分泌抗體之融合瘤細胞,並以免疫墨點法及西方轉漬法確認單株抗體。為篩選抗 CPV VP2、CDV H-N端及 CDV H-C端之單株抗體,因此以 pET32a-His融合蛋白作為篩選抗 CPV VP2及 CDV H抗體之對照組。經確認,篩選取得5株辨識 CPV VP2蛋白之單株抗體及篩選取得4株辨識 CDV H-N端之單株抗體及1株辨識 CDV H-C端之單株抗體。由免疫墨點法和西方轉漬法分析得知,抗 CPV VP2和抗 CDV H蛋白之10株單株抗體均辨識直線型抗原決定位。以 ELISA抗體亞型套組分析,結果顯示5株抗 CPV VP2蛋白之單株抗體及4株抗 CDV H蛋白之N端之單株抗體亞型皆為 IgG1。此外,1株抗 CDV H蛋白之 C端之單株抗體亞型為 IgM。以競爭性 ELISA分析單株抗體所辨識之抗原決定位,結果發現抗 CPV VP2蛋白之3株單株抗體其彼此間之競爭性在70%以上,而另外2株間競爭性則在70%以上且與前3株之競爭性在70%以下,證實此5株抗 CPV VP2之單株抗體可辨識二個不同之抗原決定位。抗 CDV H蛋白之N端之4株單株抗體彼此間之競爭性在70%以上,顯示此4株抗 CDV H蛋白之 N端之單株抗體應辨識同一抗原決定位。以 deletion mutation之方式進行抗原決定位之定位,結果顯示抗 CPV VP2之單株抗體所辨識2個抗原決定位之位置分別位於VP2蛋白之第49-89胺基酸及第90-139胺基酸。

English Abstract

Canine parvovirus (CPV) and canine distemper virus (CDV) are well-known infectious diseases of dogs.The structure of the CPV protein sheath is composed of VP1, VP2 and VP3. VP2 is the main protein and important to the immune response. CDV structure is composed of six proteins. The H protein of the virus has a very high antigenicity. In this study, the expected sizes of the VP2 of CPV and the N- and C-terminals of CDV are 83 kDa, 43 kDa and 55 kDa, respectively expressed by the prokaryotic expression system. To create hybridoma cells, BALB/c mice were immunized with the VP2 protein of CPV and the N and C terminals of CDV H protein, respectively. Antibodies secreted from hybridoma cells were screened by ELISA, immune dot, and Western blot. The monoclonal antibodies against the VP2 protein of CPV and the N and C terminals of CDV H protein were identified by using pET32a-His fusion protein as controls. There were 10 monoclonal antibodies produced in this study, including 5 anti-VP2, 4 anti-H N terminal, and 1 anti-H C terminal antibodies. Immune dot and Western blot analysis suggest that all monoclonal antibodies recognized a linear epitope. The immunoglobulin class of these 10 Mabs is determined by ELISA with a ZYMED-Mab Kit. The isotype of the anti-VP2 and the anti-H-N terminal Mabs is all IgG1. The isotype of the anti-H-C terminal is IgM. Analysis of 5 anti-VP2 and 4 anti-H N terminal Mabs by competitive ELISA suggested that 5 anti-VP2 antibodies recognized 2 different epitopes on VP2 protein of CPV and 4 anti-H N terminal antibodies recognized the same epitope on the H-N protein of CDV. Deletion mutation analysis showed that two epitopes recognized by anti-CPV VP2 monoclonal antibodies are located in the 49-89 and 90-139 amino acid residues of VP2 protein.

Topic Category 生命科學院 > 分子生物學研究所
生物農學 > 生物科學
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