Thesis (M.Sc.)--Chulalongkorn University, 2007

The broad spectrum of antimicrobial activity of the recombinant anti-lipopolysaccharide factor isoform 3 protein form Penaeus monodon (rALFPm3) was previously reported. Interestingly, the rALFPm3 is highly active against Vibrio harveyi, a shrimp pathogenic bacterium suggesting its potential use in control or prevention of the outbreak of Vibriosis in shrimp farming. Therefore, the rALFPm3 was produced in large scale in Pichia pastoris and purified by cation-exchange chromatography. The effects of extreme condition on anti-Vibrio activity of rALFPm3 were examined including salt, pH and temperature. The anti-Vibrio activity of rALFPm3 protein was reduced when the salt concentrations (1-3%) and pH (5-9) were increased. However, either incubation at various temperature (4, 30, 50 and 100oC) for 2 hours or storage at room temperature for three months did not effect the molecule integrity and its anti-Vibrio activity. We further investigated its activity in vivo in terms of neutralization and protective effect on V. harveyi challenged shrimp. At the concentration 6.25 µM, neutralization of V. harveyi (106 CFU) with rALFPm3 resulted in 100% shrimp survival. Protection of P. monodon from V. harveyi infection by pre-injected with rALFPm3 revealed a reduction of cumulative mortality from 60% to 25% comparing to the control shrimps. The effects of the rALFPm3 injection on expression levels of the representative immune genes, ALFPm3, PAP, PPA, SOD and survivin were examined in order to observe changes in shrimp immune response. The results indicated that rALFPm3 could affect several immune reactions including antimicrobial action, proPO system, antioxidant defence pathway and apoptosis but not affect the phagocytosis, Our results suggest the potential use of rALFPm3 in therapeutic and pharmaceutical applications in aquaculture.