石斑魚神經性壞死症病毒重組單源抗體的製備及表現

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

張瑞昕(Jui-Shin Chang)

指導教授 ： 齊肖琪

國立臺灣大學/生命科學院/動物學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01290

摘要

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。

關鍵字

神經壞死症病毒 ； 單源抗體 ； RACE PCR ； 重組抗體 ； GF-1 ； 被動免疫

並列摘要

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.

並列關鍵字

nervous necrosis virus ； monoclonal antibody ； RACE PCR ； recombinant antibody ； GF-1 ； passive immunity

參考文獻

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