High cell density fed-batch fermentation for the production of recombinant <Emphasis Type="Italic">E. coli</Emphasis> K-12 ghost vaccine against streptococcal disease |
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Authors: | Seung Jun Park Seok-Joo Lee Ki-Hong Kim Sung-Koo Kim |
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Institution: | (1) Aquatic Animal Disease Control Center, National Fisheries Research and Development Institute, Pusan, 619-900, Korea;(2) Pathology Division, National Fisheries Research and Development Institute, Pusan, 619-900, Korea;(3) Department of Aquatic Life Medicine, Pukyong National University, Pusan, 608-737, Korea; |
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Abstract: | The bacterial ghost system is a novel vaccine delivery method which provides versatile carrier functions for foreign antigens
with excellent natural intrinsic adjuvant properties. In this study, ghost bacteria of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM were created for mass production of a Streptococcus iniae ghost vaccine. The optimal fed-batch process for high cell density culture of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM was developed using the nutrient feeding strategy with Riesenberg defined medium. Fermentation
was conducted in four phases as follow: (1) initial batch phase, (2) fed-batch phase for high cell density culture, (3) thermal
induction phase for the formation of ghost by the expression of lysis gene E, and (4) high temperature holding phase to increase
ghost formation efficiency. The maximum ghost bacteria vaccine (GBV) was obtained from the fed-batch fermentation of 34.9
g dry cell weight (dcw)/L. The expression of antigen glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the ghost cell with
a high temperature holding phase was confirmed with outer-membrane protein fractionation using sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and Western blotting. Results indicate no damage to the expressed antigen on the ghost cell
surfaces even after the temperature was increased to 47°C for high efficiency ghost cell formation. Efficacy of the GBV was
evaluated by the challenge test in which vaccinated Olive flounder were infected with live S. iniae. The E. coli K-12 host strain, E. coli K-12/pHCE vector control, and formalin-killed cell (FKC) -treated vaccine groups showed 100, 100, and 65% cumulative mortality,
respectively. The GBV-treated groups showed 50% cumulative mortality with increased survival ratios. Hence, the immunoprotective
efficacy of GBV against S. iniae was better than that of the FKC vaccine. Therefore, the GBV is proposed as an effective vaccine in aquaculture for the prevention
of streptococcal disease. |
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