碩士 / 國立宜蘭大學 / 生物技術研究所碩士班 / 95 / Abstract
The main purposes of this research include cloning of WSSV VP28 gene and its expression under different induction conditions. The expressed recombinant proteins were used for monoclonal antibody production. In addition, the technique of absolute quantization using real-time PCR (SYBR Green I) was studied.
In PCR amplification of WSSV VP28 gene, a 629 bp DNA fragment was obtained, restriction enzyme digested, and ligated into prokaryotic expression vector pET24a. After sequence analysis confirmation for correctness, the recombinant DNA was transformed into expression host BL21 codon plus. After induction with 1mM IPTG, a 31 kDa protein was shown to be expressed and after analyzing partials of amino acid sequence by LC/MS/MS, the protein was confirmed to be WSSV VP28 (Protein coverage 27.8%). Different conditions with various temperatures of 22, 30, and 37°C, and different combinations of 0.1, 0.5, 1, and 3 mM IPTG concentrations for recombinant protein expressions were tested. It was found that regardless of the IPTG concentration, proteins expressed at 37°C were in the inclusion body state. Under lower temperature, a vast increase of soluble proteins were expressed. rVP28 soluable form proteins were purified by affinity column and used to immunize BALB/c mice. After limiting dilution and ELISA screening hybridomas, 3 different rVP28 monoclonal hybridomas were successfully produced. Western blot assay demonstrated that rVP28 monoclonal hybridoma’s cultured medium could recognized either purified rVP28 or WSSV protein.
Homogenization and extraction of WSSV infected tissue to prepare the viral DNA sample (10-1~10-8). Using both Real-time PCR and Nest-PCR technique to compare detection limite. The result showed that sensitivity of both method was similarity in viral 10-7 DNA sample. Calculation the starting quantity, is sensitive to about 89 copy number.
| Identifer | oai:union.ndltd.org:TW/095NIU07108009 |
| Date | January 2007 |
| Creators | Hsin-Hsu Tsou, 鄒行煦 |
| Contributors | Tsun-Yung Kuo, 郭村勇 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 60 |
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