Application and expression of anti-nervous necrosis virus mouse immunoglobulin G (IgG) heavy chain and light chain recombinant protein in E. coli expression system

• Main Authors: Yu-Chen Weng, 翁宇辰
• Other Authors: Chi-Yao Chang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/k4pwzq

碩士 === 輔仁大學 === 生命科學系碩士班 === 98 === Abstract Groupers are the sub-tropical and tropical fish. The importance of cultivation in Taiwan and Southeast Asia is tremendous. The groupers is fresh and delicious and the production is increasing year by year yet short in supply.Due to the high density of cultivation methods, it has led to the existence and spread of some diseases. In recent years, marine fish nervous necrosis virus has been identified as one of the most severe virus to the infantile fish. In 1986, viral nervous necrosis disease was first discovered on the Oplegnathus fasciatus of Nagasaki in Japan. Researchers today are still unable to fully understand the mechanism of NNV infection. Viral infection of groupers in southern Taiwan outbroke the first time in 1994, resulting in up to 90% infantile death. Nervous necrosis virus transmits the disease not only by a horizontal mode, but also carries on to the next generation in a vertical mode. In the treatment of nervous necrosis virus to the mother fish is then becoming very important in preventing the infection by vertical spreading to the next generation, which could result in a large number of infantile death. Ozone cleaning and disinfecting the fertilized eggs are the current method to prevent the viral nervous necrosis disease, which have reduced the incidence of the disease. Although the method could reduce the death rate, once the infantile fish is infected, it would cause a massive infection and death. Tossing the medication directly to the pond cannot be a proper way to treat the viral diseases. The main direction in researching to fight against the necrosis would be undergoing immunization. In the larval stage, fish immune system has not yet fully developed, active immunization cannot be used to prevent pathogen infection. Passive immunity at this time is the best way to solve the problem. We have produced the mouse monoclonal antibody of anti-nervous necrosis virus which has been cloned the anti-nervous necrosis virus mouse immunoglobulin G (IgG) heavy chain and light chain gene in our laboratory. In this study, we've designed the specific primers, and amplified the anti-nervous necrosis virus mouse immunoglobulin light chain and heavy chain genes by polymerase chain reaction. The amplified DNA fragments were cleaved by restriction enzymes and inserted into pET20b(+) expression vector. The obtained anti-NNV-heavy chain and anti-NNV-light chain expression plasmids were transformated into Escherichia coli strain BL21(DE3)pLysS to express anti-NNV-heavy chain recombinant protein with 469 amino acids and anti-NNV-light chain recombinant protein with 234 amino acids. The IPTG-induced recombinant proteins were purified by affinity chromatography through nickel-nitrilotriacetic acid (Ni-NTA) agarose column via 6-histidines on its C-terminal. By SDS-PAGE analysis, the molecular mass of the purified recombinant proteins were 26 kDa and 52 kDa, which is consistent with our expectation. The purpose of this study is to advance the expression and purification of the IgG heavy chain and light chain recombinant proteins, and then to experiment on the possibility of reducing the death rate by reducing the infection.