Isolation of immunoglobulin from Tilapia (Oreochromis mossambicus) and antigen characterization of Streptococcus agalactiae

• Main Authors: Hsiu-Yueh Tsai, 蔡秀月
• Other Authors: Hui-Liang Wang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/80166711559583829870

碩士 === 國立高雄師範大學 === 生物科學研究所 === 93 === Tilapia (Oreochromis mossambicus) is among one of the most important species in the aquaculture industry world wide. Streptococcus agalactiae infections have impacted significantly the local tilapia industry in Taiwan. The infection has resulted in high mortality and heavy loss every year, especially during the months with elevated water temperature. Affected fish show hemorrhage on the body surface, darkening of the body, corneal opacity, reduced appetite and splenomegaly. The S. agalactiae strain used in this study was isolated from mossambicus tilapia The pathogen is a Lancefield group B chained cocci, and present g hemolysis on sheep blood agar. The mortality was averaged 20% after a single intraperitoneal injection of bacterial suspension at the concentration of 2.5×106 cfu/ml. No mortality was observed among the survivor if am additional challenge at the same dose was given 4 weeks after the initial injection. The IgM of tilapia was first purified from crude serum by 9% PEG 6000 precipitation, and a further purification using DEAE-Sephacel column followed. The product was then analyzed by reducing and non-reducing PAGE. The results showed a single type of IgM tetramer with molecular weight at approximately 850 kDa, with 85 kDa heavy chains and 27 kDa light chains. Polyclonal antibopdy against tilapia IgM was produced by immunizing rabbit with purified tilapia IgM. A standardized ELISA assay procedure was developed using the produced rabbit antibody. A combination of 100 ml antigen (100 mg/ml), 1000X dilution of rabbit anti-tilapia IgM antibody (1 mg/ml), and 200X dilution of test fish serum each well in a standard 96-well format gave the best result. Whether naïve tilapia or infected tilapia IgM immunized rabbit could be secondary antibody. The anti-streptococcus titer peaked at 7-14 days post inoculation. Results of western blot showed two major immunogenic components in the tilapia serum, with molecular weights of 45 and 55 kDa, respectively. S. agalactiae whole bacterial cell denatured after sonication treatment appeared reaction several bands between 30 and 65 kDa. These two bands were also found 45 and 55 kDa in the sonication-disrupted bacterial proteins. The results suggest these two proteins are the major immunogens during S. agalactiae infections, and are potentially important targets for vaccine development.