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Generation of biotechnology-derived Flavobacterium columnare ghosts by PhiX174 gene E-mediated inactivation and the potential as vaccine candidates against infection in grass carp.

Zhu W, Yang G, Zhang Y, Yuan J, An L - J. Biomed. Biotechnol. (2012)

Bottom Line: Fish immunized with FCG showed significantly higher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS.Most importantly, after challenge with the parent strain G4, the relative percent survival (RPS) of fish in FCG group (70.9%) was significantly higher than FKC group (41.9%).These results showed that FCG could confer immune protection against F. columnare infection.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Animal Resistance Biology of Shandong, College of Life Sciences, Shandong Normal University, Jinan 250014, China.

ABSTRACT
Flavobacterium columnare is a bacterial pathogen causing high mortality rates for many freshwater fish species. Fish vaccination with a safe and effective vaccine is a potential approach for prevention and control of fish disease. Here, in order to produce bacterial ghost vaccine, a specific Flavobacterium lysis plasmid pBV-E-cat was constructed by cloning PhiX174 lysis gene E and the cat gene with the promoter of F. columnare into the prokaryotic expression vector pBV220. The plasmid was successfully electroporated into the strain F. columnare G4cpN22 after curing of its endogenous plasmid. F. columnare G4cpN22 ghosts (FCGs) were generated for the first time by gene E-mediated lysis, and the vaccine potential of FCG was investigated in grass carp (Ctenopharyngodon idellus) by intraperitoneal route. Fish immunized with FCG showed significantly higher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS. Most importantly, after challenge with the parent strain G4, the relative percent survival (RPS) of fish in FCG group (70.9%) was significantly higher than FKC group (41.9%). These results showed that FCG could confer immune protection against F. columnare infection. As a nonliving whole cell envelope preparation, FCG may provide an ideal alternative to pathogen-based vaccines against columnaris in aquaculture.

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Related in: MedlinePlus

Physical map of the lysis plasmid pBV E cat. Gene E, under transcriptional control of the temperature-sensitive repressor; rrBT1T2: ribosome rrnB gene providing translation stop signal terminator sequence; pro: the regulating sequence including promoter; cat: chloramphenicol acetyl transferase gene; ori: replication origin; cIts857: restraining gene of lambda bacteriophage adapted to heat induced expression.
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fig1: Physical map of the lysis plasmid pBV E cat. Gene E, under transcriptional control of the temperature-sensitive repressor; rrBT1T2: ribosome rrnB gene providing translation stop signal terminator sequence; pro: the regulating sequence including promoter; cat: chloramphenicol acetyl transferase gene; ori: replication origin; cIts857: restraining gene of lambda bacteriophage adapted to heat induced expression.

Mentions: The cat gene was amplified from the plasmid pLysS with primers Cat-F (5′-GCCGATATCATGGAGAAAAAAATC-3′, EcoRV site underlined) and Cat-R (5′- TTATCATTACGCCCCGCCCTGCCA-3′) in a standard PCR protocol. The cat gene (675 bps) was purified and cloned into pMD-18T via T/A cloning, as pT-cat. A regulating sequence including promoter was amplified from the genomic DNA of F. columnare G4 with the Pfu DNA polymerase (Fermentas) which produces blunt ended PCR products and primers Catpro-F (5′-GATAGAATAGAAAAAAGAAAATGTA-3′) and Catpro-R (5′-GGCGATTTGCCTTTTTTATAAAAT-3′). The primers were designed according to the upstream sequence of acetyl-coenzyme, a synthetase gene of F. columnare in Genbank (AY387595.2). The DNA fragment including promoter (167 bps) was subcloned into pT-cat which had been digested with EcoRV, as pT-pro-cat. The sequence including the promoter and cat gene was amplified from pT-pro-cat with the Pfu DNA polymerase and the primers Catpro-F and Cat-R and ligated into pBV-E which was predigested with ScaI. The resulting plasmid was designated as pBV-E-cat (Figure 1).


Generation of biotechnology-derived Flavobacterium columnare ghosts by PhiX174 gene E-mediated inactivation and the potential as vaccine candidates against infection in grass carp.

Zhu W, Yang G, Zhang Y, Yuan J, An L - J. Biomed. Biotechnol. (2012)

Physical map of the lysis plasmid pBV E cat. Gene E, under transcriptional control of the temperature-sensitive repressor; rrBT1T2: ribosome rrnB gene providing translation stop signal terminator sequence; pro: the regulating sequence including promoter; cat: chloramphenicol acetyl transferase gene; ori: replication origin; cIts857: restraining gene of lambda bacteriophage adapted to heat induced expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376489&req=5

fig1: Physical map of the lysis plasmid pBV E cat. Gene E, under transcriptional control of the temperature-sensitive repressor; rrBT1T2: ribosome rrnB gene providing translation stop signal terminator sequence; pro: the regulating sequence including promoter; cat: chloramphenicol acetyl transferase gene; ori: replication origin; cIts857: restraining gene of lambda bacteriophage adapted to heat induced expression.
Mentions: The cat gene was amplified from the plasmid pLysS with primers Cat-F (5′-GCCGATATCATGGAGAAAAAAATC-3′, EcoRV site underlined) and Cat-R (5′- TTATCATTACGCCCCGCCCTGCCA-3′) in a standard PCR protocol. The cat gene (675 bps) was purified and cloned into pMD-18T via T/A cloning, as pT-cat. A regulating sequence including promoter was amplified from the genomic DNA of F. columnare G4 with the Pfu DNA polymerase (Fermentas) which produces blunt ended PCR products and primers Catpro-F (5′-GATAGAATAGAAAAAAGAAAATGTA-3′) and Catpro-R (5′-GGCGATTTGCCTTTTTTATAAAAT-3′). The primers were designed according to the upstream sequence of acetyl-coenzyme, a synthetase gene of F. columnare in Genbank (AY387595.2). The DNA fragment including promoter (167 bps) was subcloned into pT-cat which had been digested with EcoRV, as pT-pro-cat. The sequence including the promoter and cat gene was amplified from pT-pro-cat with the Pfu DNA polymerase and the primers Catpro-F and Cat-R and ligated into pBV-E which was predigested with ScaI. The resulting plasmid was designated as pBV-E-cat (Figure 1).

Bottom Line: Fish immunized with FCG showed significantly higher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS.Most importantly, after challenge with the parent strain G4, the relative percent survival (RPS) of fish in FCG group (70.9%) was significantly higher than FKC group (41.9%).These results showed that FCG could confer immune protection against F. columnare infection.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Animal Resistance Biology of Shandong, College of Life Sciences, Shandong Normal University, Jinan 250014, China.

ABSTRACT
Flavobacterium columnare is a bacterial pathogen causing high mortality rates for many freshwater fish species. Fish vaccination with a safe and effective vaccine is a potential approach for prevention and control of fish disease. Here, in order to produce bacterial ghost vaccine, a specific Flavobacterium lysis plasmid pBV-E-cat was constructed by cloning PhiX174 lysis gene E and the cat gene with the promoter of F. columnare into the prokaryotic expression vector pBV220. The plasmid was successfully electroporated into the strain F. columnare G4cpN22 after curing of its endogenous plasmid. F. columnare G4cpN22 ghosts (FCGs) were generated for the first time by gene E-mediated lysis, and the vaccine potential of FCG was investigated in grass carp (Ctenopharyngodon idellus) by intraperitoneal route. Fish immunized with FCG showed significantly higher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS. Most importantly, after challenge with the parent strain G4, the relative percent survival (RPS) of fish in FCG group (70.9%) was significantly higher than FKC group (41.9%). These results showed that FCG could confer immune protection against F. columnare infection. As a nonliving whole cell envelope preparation, FCG may provide an ideal alternative to pathogen-based vaccines against columnaris in aquaculture.

Show MeSH
Related in: MedlinePlus