https://scholars.lib.ntu.edu.tw/handle/123456789/163830
標題: | 石斑魚虹彩病毒之重組蛋白抗原性分析與其抗體之應用 Analysis of antigenic proteins of grouper iridovirus and the application of antibody |
作者: | 林洋演 Lin, Yang-Yan |
關鍵字: | 石斑魚虹彩病毒;重組蛋白;多株抗體;間接三明治型ELISA;grouper iridovirus;recombinant proteins;polyclonal antibody;indirect sandwich ELISA | 公開日期: | 2007 | 摘要: | 石斑魚虹彩病毒(grouper iridovirus, GIV)基因體為140 Kb,有120個開放讀架(ORF)。以反向疫苗學(Reverse vaccinology)方式,利用小鼠及石斑魚抗血清,以西方轉漬來分析虹彩病毒具有抗原性之病毒蛋白,其結果發現大小約為60 kDa和45 kDa的病毒蛋白有明顯的訊號,參考虹彩病毒基因體的資訊,預測45 kDa蛋白質可能為主要鞘蛋白(major capsid protein, MCP),由基因GIV045R所轉譯,而60 kDa蛋白質則可能由GIV042L及GIV010R所轉譯。將這些目標基因利用大腸桿菌的表現系統進行其重組蛋白的表現與純化。之前實驗室已完成GIV010R (63 kDa),GIV042L (62 kDa)的表現與純化,另外佔有病毒顆粒最大部份病毒蛋白的主要鞘蛋白GIV045R (51 kDa),在多次全長表現遭遇困難後,予以重新設計為四個互相重疊之分段重組蛋白MCP1 (16 kDa)、MCP2 (13 kDa)、MCP3 (13 kDa)及MCP4 (11 kDa),也順利完成其表現與純化。將此6種重組蛋白利用多種動物來源之抗GIV多株抗體,以西方轉漬法來分析此重組蛋白之抗原性,發現不同動物來源的抗體辨認到的重組蛋白之抗原性各有所不同,而對於石斑魚抗血清而言,在西方墨點法的分析下,此6種重組蛋白皆具抗原性,推測所選出的6種重組蛋白對於石斑魚可能皆具有保護的潛力,而其中又以MCP3及GIV042L,訊號較為明顯,因而此兩個重組蛋白將來會是主要的研究重點。 將這些重組蛋白,以單獨以及組合的方式,混合佐劑後腹腔內注射接種石斑魚苗進行免疫,並於21天(500 ℃天)後以力價106 TCID50 ml-1之GIV病毒液進行攻毒試驗,在38天後採血進行ELISA的分析。結果發現魚苗接種各重組蛋白後,對於GIV皆存在著一定的體液免疫力,且在接種單一重組蛋白試驗之魚苗,MCP mix及GIV042L其抗GIV力價甚至高於注射不活化GIV之正對照組,此結果與西方墨點法分析的結果部份相同,顯示所有重組蛋白皆具抗原性,且可能在接種後皆有幫助魚苗誘發體液免疫力,進而抵抗GIV感染之保護效力。 在抗體的應用方面,本研究利用兔子及山羊之抗GIV多株抗體,進行間接三明治型ELISA的開發,可以用於檢測純化後的GIV,且對於NNV則無法檢測到,而當純化之GIV稀釋到16倍(約36 ng)時還可以被檢測到,顯示此分析法具有特異性及敏感性。以目前最接近野外感染的檢體,即病毒培養液來進行偵測的結果,結果當病毒培養液(原始力價為108 TCID50 ml-1)稀釋到32倍(3.125x106 TCID50 ml-1)時還可以被檢測到,進一步評估此結果,可以應用於快速免疫分析試紙之之開發。 The genome of grouper iridovirus (GIV) consists 140 Kb and encodes 120 open reading frames. The results of Western blotting showed that two proteins with 60 kDa and 45 kDa, respectively, were recognized by antibodies produced with GIV viral particles. We propose the 45-kDa protein, a deduced major capsid protein (MCP), is encoded by the gene, named GIV045R, and the 60-kDa protein might be translated from the gene, named GIV042L or GIV010R. These three genes were subcloned into pET expression vector, and their recombinant proteins were produced by E. coli expression system. Because of the difficulty to effectively express MCP, we decided to divide the MCP into four segments, named MCP1, MCP2, MCP3 and MCP4 to express. The results of Western blotting showed that all the recombinant proteins have antigenicity, but compared to the others recombinant proteins, that MCP3 and GIV042L were with strong signal. These two recombinant proteins will be the target in the future experiment. The grouper larvae were injected intraperitoneally with 2 μg of the recombinant proteins alone or combined. 21 days post-vaccination, challenged by intraperitoneally injection with 50 μl of 106 TCID50 ml-1 of GIV solution. Blood was collected and tested for ELISA after 38 days. Results of ELISA showed that all the recombinant proteins had raised titer against GIV, the group of MCP mix and GIV042L even higher than inactivated GIV-injected control group. The results showed some similarity between ELISA and Western blotting. Our results show that vaccination with the recombinant proteins may trigger the grouper larvae immune response, thus leading to protection upon live viral challeng. In this study, two polyclonal antibody against GIV from rabbit and goat were purified and used to develop an indirect sandwich ELISA for detection of GIV. The indirect sandwich ELISA could detect the purified GIV at the concentration of 36 ng and the GIV RV at the dose 3.125x106 TCID50 ml-1. Our results show that the indirect sandwich ELISA has speciality and sensitivity. These results could further be applied to immunoassay strip for the rapid diagnosis of GIV infection in grouper. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/60024 | 其他識別: | zh-TW |
顯示於: | 獸醫學系 |
檔案 | 描述 | 大小 | 格式 | |
---|---|---|---|---|
ntu-96-R94629020-1.pdf | 23.31 kB | Adobe PDF | 檢視/開啟 |
在 IR 系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。