碩士 / 國立臺灣海洋大學 / 水產養殖學系 / 97 / Viral nervous necrosis disease may cause serious economic threat to grouper culture industry. The disease may cause high mortality（80-100％）particularly in larva and juvenile. Therefore, the control and prevention of this disease is very important. Except improving culture conditions, screening broodstock fish or raising healthy fry. Using vaccine is an effective method to control the disease. The present study used E coli system to express grouper nervous necrosis virus coat protein gene for further production of recombinant coat protein as a possible candidate for efficient prophylactic subunit vaccine development.
The coat protein gene was cloned from grouper nervous necrosis virus with a fragment size of 1017 bp. The 5’ end and 3’ end were designed as Hind? site and XhoΙ site, respectively. The nucleotide sequence of the cloned gene were compared with Epinephelus coioides nervous necrosis virus truncated coat protein gene and exhibited 99％ similarity. The gene of coat protein was cloned into a pET-32a (＋) expression vector and expressed in E coli (BL21 (DE3) pLysS).
The recombinant E coli was cultured in LB/Ampcillin broth at 37℃. When the bacteria grown to OD600=0.6, 1 mM IPTG was added to induce its expression. Total protein, soluble protein and insoluble protein were collected at various time, then sodium dodecyl sulfate－polyacrylamide gel electrophoresis was used for identification. The result revealed that the use of IPTG to induce 2 hours exhibited high coat protein expression with the formation of inclusion body. The coat protein expressed was a 37 kDa protein as measured by SDS-PAGE. Furthermore, the use of 0.8 mM IPTG to induce 2 hours exhibited high coat protein expression. Metal chelate chromatography was found to be useful for recombinant protein purification. The results revealed that the 37 kDa recombinant protein could be used as a candidate for future development of subunit vaccine and application of disease diagnosis.
| Identifer | oai:union.ndltd.org:TW/097NTOU5086006 |
| Date | January 2009 |
| Creators | Yi-Sih Huang, 黃奕絲 |
| Contributors | Kuo-Kau Lee, 李國誥 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 86 |
Page generated in 0.0015 seconds