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研究生：	賴裕順 Yu-Shen Lai
論文名稱：	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
指導教授：	方剛 Fang, Kang 張繼堯 Chang, Chi-Yao
學位類別：	博士 Doctor
系所名稱：	生命科學系 Department of Life Science
論文出版年：	2001
畢業學年度：	89
語文別：	中文
中文關鍵詞：	細胞株、結病毒、單株抗體、中和試驗、免疫組織化學法、DNA疫苗
英文關鍵詞：	cell line, nodavirus, MAb, neutralization, immunohistochemistry, DNA vaccine
論文種類：	學術論文
相關次數：	點閱：187 下載：8
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從台灣南部暴發疫情的青石斑魚養殖場的病魚中，可以分離一株新的魚類神經壞死病毒(YGNNV, yellow grouper nervous necrosis virus)。從健康的青石斑魚幼苗的腦部組織，培養出一株對海水魚神經壞死病毒有感受性的細胞株為GB(grouper brain)細胞。GB細胞株是由纖維細胞及上皮細胞所組成，對海水魚神經壞死病毒的感受性，其TCID50可高達108.5。利用穿透式電子顯微鏡觀察受病毒感染的GB細胞時，發現所有成熟病毒顆粒均位於細胞質內。收集受病毒感染的GB細胞，經過二次氯化銫密度梯度離心後，可以得到純化的神經壞死病毒，病毒在PTA負染色後，在電子顯微鏡觀察下，呈現直徑約25-30 nm，形狀類似圓形的二十面體病毒顆粒。這個結果證明我們培養的青石斑魚GB細胞株適合用來研究水海魚神經壞死病毒。
利用從GB細胞株純化而來的神經壞死病毒當作免疫抗原，注射到老鼠腹腔內，來誘導老鼠的免疫反應，接著進一步將有免疫反應的老鼠脾臟細胞取出，製備抗神經壞死病毒的單株抗體。得到的融合瘤細胞經過篩選後，分析其中十個最佳中和神經壞死病毒感染力價之單株抗體，最高可達106.5中和指數，這些單株抗體都屬於IgG的類型，而且都含有一個  輕鏈。經過西方點墨法的分析，證明所有的單株抗體都結合到病毒42 kDa 的外鞘蛋白上。利用單株抗體來進行免疫組織化學分析法，結果證實病毒感染分布主要集中在視網膜神經，腦部，以及脊髓神經。這些結果說明我們製備的抗神經壞死病毒的單株抗體，不但可以用來診斷並且可用來中和神經壞死病毒的感染能力。
將抑制神經壞死病毒感染效價最好的老鼠單株抗體融合瘤細胞MAb-18的mRNA分離純化出來，建立cDNA基因庫，並且將表現單株抗體的重鏈以及輕鏈的基因分離出來。將此重鏈及輕鏈基因單獨或兩者構築在以CMV為啟動子的表現載體上(pCMV-NNV-18H, pCMV-NNV-18L, pCMV-NNV-18HL)，然後載體轉殖入GB細胞株內，以免疫細胞化學法可以顯示，單獨或同時轉殖重鏈及輕鏈基因的GB細胞皆可表現老鼠的抗體基因。此轉殖表現之抗體並可自GB細胞分泌至培養基中，且在同時表現重鏈及輕鏈時，彼此間可以雙硫鍵結成IgG。病毒中和實驗證實細胞內生性抗體以及分泌性抗體都具有中和神經壞死病毒感染力價的能力，且此內生性抗體可以抑制病毒之繁殖能力。實驗結果證明所使用的這個方法不僅可以用來生產良好的抗神經壞死病毒DNA疫苗，更可以進一步來製造抗體基因轉殖魚種，對抗魚類致病原的感染。

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.

Contents
                                                                                                                            page
中文摘要                                                       1
Abstract                                                       3
Introduction                                                 5
Materials and methods                                       10
1.Light microscopy                                          11
2.RNA extraction                                            11
3.Reverse transcription and PCR amplification               11
4.Cloning and sequencing of PCR products                    12
5.Sequence alignment                                        13
6.Primary culture of grouper brain (GB)                     14
7.Subculture and maintenance                                14
8.Chromosome preparation                                    15
9.Growth studies                                            15
10.Viral susceptibility of GB cell                          16
11.Effect of temperature on YGNNV replication in GB cells   17
12.Isolation and purification of YGNNV                      17
13.Electron microscopy                                      18
14.Viral antigen preparation                                19
15.Preparation of monoclonal antibody(MAb)                  19
16.Enzymelinked immunosorbent assay(ELISA)for MAb screening 20
17.Neutralization test of MAb to YGNNV in GB cell           21
18.Isotype determination                                    21
19.SDS-PAGE and Western blot analysis ofthe viral          polypeptides                                                  22
20.Virus coat protein synthesis in infected GB cells        22
21.Immunohistochemistry                                     23
22.Construction of cDNA                                     23
23.Probeation                                               24
24.Colony hybridization                                     24
25.Expression vectors construction                          25
26.Transfection of GB cells                                 26
27.RT-PCR detection of MAb-18 gene expression               26
28.Immunocytochemistry                                      27
29.Western blot detection for secretary MAb-18              27
30.ELISA detection for secretary MAb-18                     28
31.Neutralization assay of intra- and extracellular antibodies against YGNNV                                      29
32. Virus susceptibility of transfected GB cells            29
Results                                                       31
1.Histopathology                                            32
2.RT–PCR amplification and sequences of YGNNV coat protein gene                                                          32
3.Primary and subculture of GB cells                        33
4.Chromosome analysis                                       33
5.Growth studies                                            33
6.Virus susceptibility of GB cells                          34
7.Effect of temperature on YGNNV replication in GB cells    34
8.Electron-microscope observation                           34
9.Purification of the virus                                 35
10.ELISA for MAb screening                                  35
11.Neutralization assay                                     36
12.Isotype determination                                    36
13.SDS-PAGE and Western blot analysis of the viral polypeptides                                                  36
14.Time course synthesis of YGNNV polypeptides in infected GB cells                                                         37
15.Immunohistochemistry of infected grouper                 37
16.Isolation and cloning of MAb- 18 heavy and light chains  38
17.Expression of MAb-18 genes in GB cells                   39
18.Immunocytochemistry                                      39
19.Secretion of antibody by GB cells                        39
20.Binding activity of intra- and extracellular recombinant antibodies                                                    40
21.Virus susceptibility of the intracellularly immunized GB cells                                                         40
Discussion                                                  41
References                                                  48
Table                                                       59
Table 1 Nucleotide and amino acid sequence similarities of the T2 regions of YGNNV and six other fish nodavirus strains      60
Figures                                                     61
Figure 1.Histological sections of normal and YGNNV-infected grouper tissues stained with haematoxylin and eosin.          62
Figure 2.Physical map and agarose gel electropherogram of PCR products of the YGNNV coat protein gene.                      63
Figure 3.Multiple alignment of T2 nucleotide sequences from seven piscine nodaviruses.                                    64
Figure 4.Phase contrast micrograph and chromosomes giemsa-stained of GB cells                                           65
Figure 5.Influence of incubation temperature and different concentrations of FBS on growth of GB cells.                  66
Figure 6.Cytopathology of GB cells infected with YGNNV and effect of different temperatures on YGNNV replication in GB cells.                                                        67
Figure 7.Transmission electron micrograph of YGNNV infected GB cells and electron micrograph of negatively stained purified virus particles.                                              68
Figure 8.Neutralization activity of MAbs to YGNNV in GB cells.                                                        69
Figure 9.SDS-PAGE and Western blot analysis of YGNNV coat protein.                                                      70
Figure 10.Virus coat protein synthesis in YGNNV-infected GB cells.                                                        71
Figure 11.Immunohistochemistry of paraffin sections of normal and nodavirus-infected grouper tissues.                       72
Figure 12.Schematic representation and the deduced amino acid sequence of the multiple domains of MAb-18H.                  74
Figure 13.Schematic representation and the deduced amino acid sequence of the multiple domains of MAb-18L.                  75
Figure 14.Schematic representation of construction of pCMV-NNV 18L, pCMV-NNV 18H and pCMV-NNV 18HL expression vectors.       76
Figure 15.Detection of IgG transcripts in GB cells, transfected with different plasmid constructs, by RT-PCR.                 77
Figure 16.Localization of intracellularly expressed light or heavy chains of trans-fected GB cells by immunocytochemistry. 78
Figure 17.Western blot analysis of extracellular antibody of transfected GB cells under reducing or non-reducing conditions79
Figure 18.Neutralization assay of extra- and intracellular antibodies from transfected GB cells with pCMV-NNV 18H, pCMV-NNV 18L and pCMV-NNV 18HL vector, against YGNNV.              80
Figure 19.Propagation of YGNNV in intracellularly immunized GB cells with the pEGFP-N1, pCMV-NNV 18H, pCMV-NNV 18L and pCMV-NNV 18HL vectors.                                             81
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