碩士 / 國立臺灣大學 / 微生物與生化學研究所 / 93 / The material of this study is the Cobia(Rachycentron canadum) which were infected by iridovirus and had lymphocystis syndrome. We isolated the genomic DNA of several organs and tissues and designed specific primers to detect and amplify the major capsid protein gene mcp by polymerase chain reaction (PCR). After sequencing, the total length of mcp is 1295 base pairs. Then, we used the software Bioedit to analyze the restriction map and simulate its translation product, MCP. The number of amino acid composing MCP is 431, which suggested that the molecular weight should be between 45 to 50 kD.
We isolated viral particles from infected organs and tissues by blending method and emulsified viral particles to immune Balb/c female mouse in order to produce monoclonal antibody (mAb). Immunization methods are traditional method and RIMMS. Then the spleen cells of immuned Balb/c mouse were collected and fused with NS-1 myeloma cells by PEG-inducing method. The fused cells were then selected in HAT medium. The hybridomas, which could secrete anti-MCP antibodies, were screened with enzyme-linked immunosorbant assay (ELISA). Then the hybridomas were subcloned by limit dilution. Two hybridomas producing anti-MCP mAb were obtained and designated as 3C6 and 2A10. The isotypes of 3C6 and 2A10 were IgG1 and IgG2a as heavy chain and same κ as light chain. 3C6 hybridoma secreted higher titer of anti-MCP antibodies and had better growth conditions than 2A10 hybridoma, thus we chose 3C6 hybridoma to produce anti-MCP mAb. Then we purified antibodies by following methods: ammonium sulfate participation, ion-exchange (DEAE) chromatography, and Protein-G affinity chromatography. SDS-PAGE and ELISA were used to analyzed purified products.
Finally, we cloned mcp gene into pQE31 expression vectors and expressed MCP by E. coli DH5α. The recombinant MCP protein was purified easily because it carried 6x His-tag on N-terminal. After Ni-NTA affinity chromatography, pure MCP protein was collected, and its molecular weight was about 48 kD. Western blotting showed that recombinant MCP would interact with 3C6 mAb specifically.
With indirect ELISA, purified 3C6 antibodies were diluted to 104 times to interact with different concentrations of MCP protein. The MCP concentration ranged form 0.5 to 5 μg/mL showed a better linear relationship, and the detection limit was 0.5 μg/mL.
In summary, we produced antibodies which interacted with major capsid protein of iridovirus that were isolated from cobia with lymphocystis syndrome. We hope that the mAb produced in this study can apply in virus detection. Otherwise, the recombinant MCP also has a potential to develop as vaccines.
| Identifer | oai:union.ndltd.org:TW/093NTU05381028 |
| Date | January 2005 |
| Creators | Yang-Wei Liu, 劉楊威 |
| Contributors | 陳建源 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 122 |
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