以生物製劑防治蝦類白點症病毒水平傳染

Abstract

蝦類白點症病毒 (White spot syndrome virus, WSSV) 在九零年代初期首次出現於亞洲，由於它可感染的甲殼類宿主種類相當廣泛，且在感染後七到十天便可造成養殖蝦近百分之百的死亡，因此疫情迅速擴散至全球，所到之處均造成蝦類繁養殖場極大的的損失，迄今仍是危害全球養蝦產業的重要疾病之一。基於蝦類養殖是台灣重要的產業，民間公司,Transcending Biotechnologies, Inc.,成功研發出能有效中和白點症病毒的單株抗體(AP-1)。過去數年中該公司與國立臺灣海洋大學和國立台灣大學合作之田間實驗，證實以AP-1處理帶有白點症病毒的草蝦種蝦及幼苗，可成功生產出檢測無病毒的蝦苗，達到預防垂直傳染的目標。而無病毒的蝦苗，仍無法克服環境中帶原生物所導致的水平傳染，必須要將之杜絕才能成功收穫成蝦。由於成本考量在養殖產業是重要的環節，如果AP-1要擴大使用在防止水平傳染，就必需要改變傳統以哺乳類細胞生產之方式，從而降低其生產成本才能有實用價值，方可真正運用在產業上。 乳酸菌是國際上認可對人體無害且有益的微生物(Generally Recognized as Safe, GARS)，並且在養殖產業普遍都認為可以增加養殖物種的產量。近年來發展之乳酸菌蛋白質表現系統(Nisin Controlled gene Expression system, NICE)不再使用抗生素作為篩選方法，所生產之抗病毒生物製劑較沒有安全疑慮。但細菌表現系統無法表現完整抗體，所以將以單鍊抗體(Single-chain variable fragment, scFv)型式表現AP-1。 本實驗是建立在此背景下，目的是利用微生物蛋白質表現系統生產低成本的抗病毒生物製劑。 實驗結果顯示，scFv-AP-1(sAP-1)能在乳酸菌蛋白質表現系統中成功表現重組蛋白，但沒有如預期之方式分泌至培養液中，所以轉而利用較為經濟並可由胞質間隙中萃取蛋白質之大腸桿菌來確認sAP-1功能及保護力。表現之sAP-1在ELISA實驗中可與WSSV VP28蛋白結合，並與AP-1有 VP28之結合相互競爭現象，sAP-1和AP-1應具有相同之結合點。攻毒試驗結果顯示sAP-1保護效果達到存活率60%，但在real-time PCR檢測其病毒結果中，死亡及存活皆可偵測到高病毒量，推測雖具有保護效果但如果無法完全抑制病毒，後期還是有病毒潛伏的可能性；因此，現場使用方式與劑量的控制將是下一階段試驗之重點。

White spot syndrome virus (WSSV) was first emerged in Asia in the early 1990’s. It has a wide range of hosts among crustaceans and causes up to 100% mortality within 7 to 10 days in cultured shrimps. It was spread out all over the world rapidly. Until now, white spot syndrome has become one of the most serious viral diseases of cultivated shrimp and causes considerable economic losses to the shrimp farming industry worldwide. The monoclonal antibody AP-1 was developed by a biotech company, Transcending Biotechnologies, Inc. (TBI), which was demonstrated that could neutralize WSSV in laboratories. In the past years, TBI, National Taiwan Ocean University, and National Taiwan University have collaborated for field tests and concluded that the broodstocks and larval of Penaeus monodon that were treated with AP-1 had no WSSV been detected. Although the vertical transmission of WSSV can be prevented by AP-1 treatment, the WSSV is still able to infect shrimp through horizontal transmission. To ensure the harvest of shrimp, the horizontal transmission of WSSV has to be solved. Since a relatively huge amount of AP-1 needed to prevent horizontal transmitted WSSV, the production cost needs to be reduced. The production of AP-1 from microbial expression systems were therefore selected and tested. Lactic acid bacteria (LAB) are internationally accepted as “Generally Recognized as Safe” (GARS) probiotics that are harmless to human and are generally believed can increase the production of cultured animals. Additionally, in the recent years, the LAB based Nisin Controlled Gene Expression system (NICE) has been developed which does not require antibiotic selection and has less safty concerns. Because bacteria usually are difficult to express conformationally correct antibody, the AP-1 was reconstructed to a single chain variable fragment (scFv) form. The goal of this study was to express the anti-WSSV biologics scFv-AP-1 (sAP-1) by NCIE or other microbial expression systems. The results showed that sAP-1 can be expressed in NICE system, but it does not secret to medium and difficult to be extracted from cells for further purification. The expression systems were then switched to E.coli expression systems so that sAP-1 could be evaluated for its binding reactivity and biological function. The binding activity of E.coli expressed sAP-1 to VP28 was confirmed through ELISA and its binding site was further verified by competition assay with AP-1 as competitor. In the viral challenge experiments, Cherax quadricarinatus received PBS or unrelated protein plus WSSV were all dead at day 6 and sAP-1 treated group had 60% survival rate at day 12 which was less than control group without WSSV and AP-1 treated group’s 100% protection. Some of survived shrimps in sAP-1 group still remained high levels of WSSV by real-time PCR assay. The data clearly indicates that sAP-1 can protect shrimp from WSSV infection but the dosage and application procedure have to be determined before having the maximal protection.