The simple fed-batch fermentation was carried out to produce E. coli XL1-Blue/pHCE-InaN-GAPDH Ghost 37 SDM as a ghost bacterial vaccine (GBV). The fermentation was carried out in four phases of batch fermentation (phase I), fed-batch fermentation with intermittent feeding strategy (phase 2), thermal induction by temperature increase to 42 degrees C for the expression of lysis E gene (GBV formation, phase 3) and high temperature holding phase to increase the efficiency of GBV formation (phase 4). After the high temperature holding phase at 47 degrees C, efficiency of the GBV formation reached 99.7% with the culture OD(600) of 57.9. The maximum GBV of 22 g dcw/l was obtained. The protective efficacy of GBV was determined by a challenge test to immunized olive flounder using live Streptococcus iniae. In 14 days of challenge test, the positive and E. coli strain control groups showed 100% Cumulative mortalities. Test groups immunized by formalin killed cell (FKC) vaccine, GBV with 42 degrees C and 47 degrees C heat shock showed 66%. 54% and 54% Of Cumulative mortalities, respectively. These results suggest that GBV showed the effectiveness for the protection from the streptococcal infection and had higher potential to induce protective antibodies than FKC vaccine. (C) 2009 Elsevier Ltd. All rights reserved.