青石斑魚神經壞死病毒特性分析及其免疫研究

Characterization of nervous necrosis virus (NNV) isolated from yellow grouper, Epinephelus awoara and its immunity study

賴裕順(Yu-Shen Lai)

指導教授 ： 方 剛 ； 張 繼 堯

國立臺灣師範大學/理學院/生物學系/博士(2000年)

摘要

無資料

關鍵字

細胞株 ； 結病毒 ； 單株抗體 ； 中和試驗 ； 免疫組織化學法 ； DNA疫苗

並列摘要

Fish nodavirus was isolated from the diseased yellow grouper, Epinephelus awoara, larvae as yellow grouper nervous necrosis virus (YGNNV). A new nodavirus-susceptible cell line, derived from grouper brain (GB) was also established and characterized. The GB cell line is made of fibroblast-like cells and epithelioid cells. The cell line yielded a high titre of up to 108.5 (TCID50 /ml). Under electron microscope, aggregation and replication of the virus particles were observed in the cytoplasm of the brain cells. The virus were purified to homogeneity by caesium chloride gradient centrifugation. The purified virus particles were 25-30 nm in diameter as determined by electron microscopy. The result showed that GB cell line is a useful tool for studying fish nodaviruses.
YGNNV-specific monoclonal antibodies (MAbs) were also produced. Ten of the clone, showed high neutralization index up to 106.5. These MAbs belonged to the IgG isotype with a κ light chain that recognized the 42 kDa coat protein of YGNNV as shown by Western blot analysis. Immunohistochemical study showed that the viral signals were co-located with pathological lesions observed in retina, brain and spinal cord. Using these MAbs that we ideal for the diagnosis of YGNNV infection.
Further, the genes encoding heavy and light chains of the YGNNV-specific MAb-18 were isolated from cDNA library of the hybridoma. Three expression constructs, pCMV-NNV-18L, pCMV-NNV-18H, and pCMV-NNV-18HL encoding light, heavy and the combined heavy and light chains, respectively, were made and expressed in GB cells. Intracellular expression of the heavy and light chains of MAb-18 was localized by immunocytochemical staining. Western blotting results indicated that the expressed antibody secreted into the medium with correct folding. These extracellular and intracellular antibodies reacted and neutralized well the coat protein of YGNNV. The intracellular antibody was found to reduce the NNV titre significantly level. This approach can be applied for DNA vaccines, and transgenic fish that are expected resistant to fish pathogens.

並列關鍵字

cell line ； nodavirus ； MAb ； neutralization ； immunohistochemistry ； DNA vaccine

參考文獻

1. Arimoto M., Mushiake K., Mizuta Y., Nakai T., Muroga K., Furusawa I. Detection of striped jack nervous necrosis virus (SJNNV) by enzyme-linked immunosorbant assay (ELISA). Fish Pathology 1992; 27:191–195.

2. Biocca S, Neuberger MS, Cattaneo A. Expression and targeting of intracellular antibodies in mammalian cells. EMBO J 1990; 9(1):101-108.

3. Biooca S, Cattaneo A. Intracellular immunization: antibody targeting to subcellular compartments. Trends In Cell Biology 1995; 5:248-252

4. Bloch B., Gravningen K., Larsen J.L. Encephalomyelitis among turbot associated with a picornavirus-like agent. Diseases of Aquatic Organisms 1991; 10:65–70.

5. Boonyaratpalin S., Supamattaya K., Kasornchandra J., Hoffmann R.W. Picorna-like virus associated with mortality and a spongious encephalopathy in grouper Epinephelus malabaricus. Diseases of Aquatic Organisms 1996; 26:75–80.