Development and in vitro expression of the recombinant monoclonal antibody against grouper nervous necrosis virus

海水魚苗繁養殖是台灣重要的經濟產業之一，而石斑魚繁養殖尤具經濟價值。石斑魚苗的飼育期間深受病毒性神經壞死症(Viral Nervous Necrosis Disease；VNN)之威脅造成嚴重的經濟損失。造成VNN的致病原為神經壞死症病毒（NNV），分類上屬於野田病毒科（Nodaviridae），β野田病毒屬（betanodavirus），具有兩段單股正意核醣核苷酸（positive-sense RNA）。石斑魚苗免疫系統至孵化後約20-30天左右成熟，才適合進行主動免疫，但免疫後仍需要3-4週才能誘發足夠抗體對抗病毒感染。然而，NNV對於孵化後一個月之內的幼苗仍具有很高的威脅性，因此主動免疫方式仍無法保護此階段幼苗。故擬使用被動免疫策略來補足防疫上的空窗期。本實驗室先前已建立五株對NNV具有中和力價之單源抗體，其中代號9D之單源抗體具高中和力價且可辨識線性抗原決定位（Epitope）。因此本實驗自老鼠融合瘤細胞株（9D）選殖單源抗體的輕鏈及重鏈基因，並以5’及3’ RACE完成兩基因之全長定序，再將基因轉至合適的表現載體，建構之表現載體共轉染（co-transfection）於石斑魚鰭細胞株（GF-1）中，利用RT-PCR確認重組抗體mRNA表現；用Western blot確認有單源抗體蛋白質之表現與分泌，並確認重組抗體9D可結合NNV鞘蛋白質。未來此重組單源抗體將有潛力應用於石斑幼苗的被動免疫，以降低NNV對石斑魚苗的威脅性。The aquaculture production of marine fish is an important economic industry in Taiwan. One of the highly value marine fish is groupers. However, the occurrence of viral nervous necrosis disease persists as a stumbling block that besets sustainable production in grouper larvae. Nervous necrosis virus (NNV) is the causative agent of mass mortality of cultured grouper at larval stage. It belongs the betanodavirus of Nodaviridae, with 2 segments of single strand positive sense RNA. The immune system of grouper larvae are not mature enough to be immunized until 20-30 days post hatchery (d.p.h.). Therefore, the larvae are very sensitive to NNV infection within 30 d.p.h. Passive immunization was therefore an alternative prophylaxis strategy against NNV infection at this stage. Five NNV neutralization monoclonal antibodies (mAb) are developed in our previous study. The mAb 9D exhibits high neutralizing activity and recognizes linear epitope of capsid protein. In the present study, the heavy and light chain genes of hybridoma cells of mAb 9D were cloned. The full length of these two genes were determined by 5’ and 3’ RACE PCR, and then inserted into the expression vector, respectively. The constructed vectors were transfected into GF-1 cells. The mRNA of heavy chain and light chain genes were detected by RT-PCR, and the antibody proteins expressed in the transfected cells were detected by western blot. Moreover, the expressed mAb 9D in the culture supernatant of transfected cells was confirmed to recognize NNV capsid protein by western blot. In the future, the constructed mAb may be applied in the passive immunity of grouper larvae in order to decrease threaten of VNN disease.目錄................................................................................................................................I文摘要…………………………………………………………………….....…....VI文摘要....................................................................................................................VII獻回顧…………………………………………………………….…......................1. 石斑魚生物特性與養殖現況..........................................................................1. 神經壞死症病毒..............................................................................................2.1病史及病癥.............................................................................................2.2特性及分類地位.....................................................................................3.3地理分佈與宿主.....................................................................................4.4流行病學.................................................................................................6.5病程及在宿主體內之分佈.....................................................................7.6診斷方法.................................................................................................8.7防疫.......................................................................................................10. 魚類免疫系統................................................................................................13.1魚類免疫系統簡介...............................................................................13.2魚類免疫系統的發生...........................................................................14.利用免疫方式之病毒防治策略......................................................................15.1主動免疫...............................................................................................15.2被動免疫...............................................................................................16.抗體於防疫策略中的應用..............................................................................17料方法.....................................................................................................................19. 選殖老鼠融合瘤細胞9D之單源抗體基因..................................................19.1培養小鼠融合瘤細胞...........................................................................19.2萃取老鼠融合瘤細胞之RNA...............................................................19.3利用反轉錄酶製備cDNA.....................................................................19.4單源抗體重鏈及輕鏈之聚合酶鏈鎖反應...........................................20.5 膠體純化..............................................................................................20.6 DNA接合..............................................................................................21.7 大腸桿菌勝任細胞製作......................................................................21.8質體轉型...............................................................................................21.9菌落培養及PCR檢測...........................................................................22.10核酸序列比對與分析.........................................................................22. 以RACE PCR方式定序9D抗體基因..........................................................22.1 重鏈部分..............................................................................................22.1.1 5’ RACE PCR............................................................................22.1.2 3’RACE PCR.............................................................................24.1.3序列合成....................................................................................24.2輕鏈部分...............................................................................................24.2.1 5’ RACE PCR............................................................................25.2.2 3’RACE PCR.............................................................................25.2.3序列合成....................................................................................26. 產生9D-Hμ基因重組載體............................................................................26.1 增幅9D-Hμ完整基因片段..................................................................26.2 質體純化..............................................................................................27.3限制酶截切...........................................................................................27.4膠體純化、DNA接合、質體轉型..........................................................28.5 菌落培養、限制酶截切檢測..................................................................28. 產生9D-Lκ基因重組載體............................................................................28. pN2-HT、pN2-LT質體共轉染........................................................................29.1培養石斑魚鰭細胞株（GF-1）...............................................................29.2細胞轉染...............................................................................................30.3 篩選共轉染後的GF-1細胞.................................................................30. 共轉染GF-1細胞重鏈、輕鏈表現測試.........................................................30.1 重鏈、輕鏈mRNA表現測試...............................................................30.2 重鏈、輕鏈蛋白質表現測試................................................................31.2.1 細胞蛋白質粗萃取...................................................................31.2.2 蛋白質膠體電泳.......................................................................31.2.3 蛋白質轉印...............................................................................32.2.4 西方點漬...................................................................................32.3 重組抗體辨識抗原測試......................................................................32果.............................................................................................................................33. 選殖老鼠融合瘤細胞9D之單元抗體基因..................................................33. 以RACE PCR方式定序9D抗體基因..........................................................33. 建立9D單源抗體重鏈基因重組載體（9D-Hμ）...........................................33. 建立9D單源抗體重鏈基因重組載體（9D-Lκ）............................................34. 共轉染GF-1細胞重組抗體重鏈、輕鏈表現測試.........................................34.1 mRNA表現測試...................................................................................34.2 蛋白質表現測試..................................................................................34.3轉染載體細胞產生之重組抗體能辨識NNV病毒..............................35論.............................................................................................................................36. 定序9D抗體基因.........................................................................................36. 建立9D單源抗體重鏈（9D-Hμ）及輕鏈（9D-Lκ）基因重組載體................37. 共轉染GF-1細胞重組抗體重鏈、輕鏈表現測試.........................................37. 未來展望........................................................................................................39考文獻…………………………………………………………………….……....41目錄1..............................................................................................................................56目錄1：選殖之單源抗體部分基因序列確認.................................................................572：單源抗體重鏈之全長序列.................................................................................583：單源抗體輕鏈之全長序列.......................................................................59 4：單元抗體重鏈與輕鏈全長基因電泳圖...........................................................60 5：以限制酶截切確認抗體序列接合方向...........................................................61 6：共轉染GF-1細胞株重鏈、輕鏈mRNA表現電泳結果...................................62 7：重組抗體質體轉染GF-1細胞蛋白質萃取液中重鏈及輕鏈表現結果..........63 8：重組抗體質體轉染GF-1細胞培養液中重鏈及輕鏈表現結果......................64 9：9D重組抗體功能性測試結果..........................................................................65錄.............................................................................................................................66器.....................................................................................................................66品試劑.............................................................................................................66物反應試劑組.................................................................................................72驗載體.............................................................................................................7