使用重組蛋白製造對 HPV、IHHNV 和TSV殼蛋白有專一性之單株抗體

Abstract

根據聯合國糧食及農業組織(FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS，FAO)的數據指出，蝦養殖產業為一個能夠提供高營養價值的食物來源，也能為發展中國家提供經濟收入的一重要之產業。但近年來，因為病毒問題，使其遭受巨大之損失。因此若是能有效且儘早偵測到病毒，可以針對所面臨之狀況，提早作出對應，以降低損失。 本研究從NCBI database資料中，分別在肝胰小病毒 (hepatopancreatic parvovirus, HPV)、傳染性皮下及造血組織壞死病毒 (infectious hypodermal and hematopoietic necrosis virus, IHHNV) 和桃拉病毒 (taura syndrome virus, TSV) 等三種常見之蝦病毒中，各選出一段殼蛋白基因，利用重組蛋白的方式，在大腸桿菌中大量表現。並利用親和性管柱純化，將純化所得之蛋白質當作抗原注射至老鼠體內誘導，以期發展出針對這些蛋白質具有專一性的單或多株抗體。 目前實驗已可以成功地利用大腸桿菌，大量表現HPV、TSV及IHHNV的重組蛋白，並利用重組蛋白上所帶的親和性標記進行純化，再以這個純化的重組蛋白，在BALB/c老鼠體內引發免疫反應，經由ELISA及西方點墨實驗證實，可以免疫出對重組蛋白具有反應的單株抗體。之後結合融合瘤技術發展單株抗體，在HPV方面，總共得到2株細胞株。在西方點墨實驗中，均可以辦別到HPV重組蛋白。在蝦體的檢驗中，使用3-24這株單株抗體進行西方點墨實驗，其初步結果與病毒檢測試劑 (PCR diagnosis) 所得結果相符。在IHHNV目前篩選到3-62這株細胞株，可辯認到IHHNV重組蛋白。經由初步結果顯示，証實所發展的單株抗體，具有發展成檢驗試劑的可能性。

As the FAO (FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS) reported, the aquaculture of shrimp is a very important industry. It can support high nutritional food and provide the income for the developing nation. Because of the viral disease, this industry suffers huge loss recently. Therefore, if the detection of virus can be effective as soon as possible, we may prevent the diseases from the shrimp. In this study, we refer to the NCBI database to design primers which are used for amplification of the gene fragment of hepatopancreatic parvovirus (HPV)、infectious hypodermal and hematopoietic necrosis virus (IHHNV) and taura syndrome virus (TSV). These gene-encoding structural coat proteins were cloned into expression vector and transformed into E. coli. The objective was to produce recombinant coat protein with a 6-histidine tag. After induction, the recombinant proteins were produced, purified by Nickel column and used for immunization of BABL/c mice for polyclonal antibody production. The mouse antiserum showed specific immunoreactivity to the recombinant protein as verified by ELISA and Western blot. In HPV, the western blot data indicated that two monoclonal antibodies against the HPV recombinant protein were constructed. The dectection of shrimps with monoclonal antibody 3-24 strain exhibited parallel result as compared with that of PCR diagnosis. In IHHNV, 3-62 strain showed immunoreactivity against the IHHNV recombinant protein or coat protein purified from the IHHNV infected shrimp.