Establishment of a continuous cell line derived from barramundi (Lates calcarifer ) and its applications

• Main Authors: Jia-Rong Chang, 張家榮
• Other Authors: Tsun-Yung Kuo
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/52636999813684799231

碩士 === 國立宜蘭大學 === 生物技術研究所碩士班 === 95 === Lates calcarifer (bloch) belongs to the Lates genus, Centropomidae family, Perciformes order and Osteichthyes class. This tropical and sub-tropical fish is located in the coastal brackish water throughout Okinawa to the Indian Ocean. Lates calcarifer is massively cultured in Southeastern Asia and Southern Taiwan. Nervous Necrosis Virus (NNV), a member of the Betanodaviridae, is an icosahedral, non-enveloped RNA virus 25-30 nm in diameter. NNV mainly infects juveniles, destroying the nerves of the brain, leading to abnormal swimming and high mortality of infected fish, causing devastating economic impact to the aquaculture industry. Since the larvae of Lates calcarifier are highly susceptible to NNV, this research study developed primary and continuous cell lines from the liver, kidney, and the gas bladder of bloch. The liver and kidney cells were propagated over 90 passages while the gas bladder cell line was propagated over 150 passages, maintained in 5% FBS, L-15 medium, and grew quickly at both 28°C and 32°C. Epinephelus lanceolatus NNV was first used to test these different cell lines for susceptibility and results showed that Lates calcarifer gas bladder (LCGB) was highly susceptible. Then, wild type Plectropomus leopardus Nervous Necrosis Virus (PLNNV) was replicated in LCGB and the viral titer was 107 TCID50/mL. Large quantities of PLNNV was grown in LCGB, collected and purified, serving as the antigen for anti-NNV monoclonal antibodies production in BALB/c mice. After limiting dilution and screening, 3 different hybridomas secreting monoclonal antibodies against NNV were successfully produced. The 3 monoclonal antibodies were confirmed by Western blot and IPMA against purified NNV. Subsequently, cloning of PLNNV capsid gene and expression of recombinant proteins in E. coli was done in pQE30 vector and M15 host. A Western blot of the monoclonal antibodies against the recombinant protein proved all 3 monoclonal antibodies produced in this study were against the NNV capsid protein. These monoclonal antibodies are important tools to have for further researches on Nervous Necrosis Virus.