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Article: A method to generate recombinant Salmonella typhi Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy

TitleA method to generate recombinant Salmonella typhi Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy
Authors
KeywordsRecombineering Salmonella vaccine
Issue Date2011
PublisherSpringer. The Journal's web site is located at http://link.springer.de/link/service/journals/00253/index.htm
Citation
Applied Microbiology And Biotechnology, 2011, v. 91 n. 1, p. 177-188 How to Cite?
Abstract
Live attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated 'targeting cassettes' that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking 'arms' of sequence (ca. 1,000 bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains. © 2011 Springer-Verlag.
Persistent Identifierhttp://hdl.handle.net/10722/138958
ISSN
2013 Impact Factor: 3.811
ISI Accession Number ID
Funding AgencyGrant Number
RFCID
NSFC
Hong Kong Research Grants CouncilN_HKU 719/08
CRCG
Funding Information:

This work was supported by RFCID grants to JDH, a grant from NSFC and the Hong Kong Research Grants Council (N_HKU 719/08) to JDH and ES, and a grant from CRCG Seed Funding Programme for Applied Research to JDH.

References
Grants

 

DC FieldValueLanguage
dc.contributor.authorYu, Ben_HK
dc.contributor.authorYang, Men_HK
dc.contributor.authorWong, HYBen_HK
dc.contributor.authorWatt, RMen_HK
dc.contributor.authorSong, Een_HK
dc.contributor.authorZheng, BJen_HK
dc.contributor.authorYuen, KYen_HK
dc.contributor.authorHuang, JDen_HK
dc.date.accessioned2011-09-23T05:43:07Z-
dc.date.available2011-09-23T05:43:07Z-
dc.date.issued2011en_HK
dc.identifier.citationApplied Microbiology And Biotechnology, 2011, v. 91 n. 1, p. 177-188en_HK
dc.identifier.issn0175-7598en_HK
dc.identifier.urihttp://hdl.handle.net/10722/138958-
dc.description.abstractLive attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated 'targeting cassettes' that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking 'arms' of sequence (ca. 1,000 bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains. © 2011 Springer-Verlag.en_HK
dc.languageengen_US
dc.publisherSpringer. The Journal's web site is located at http://link.springer.de/link/service/journals/00253/index.htmen_HK
dc.relation.ispartofApplied Microbiology and Biotechnologyen_HK
dc.rightsThe original publication is available at www.springerlink.com-
dc.subjectRecombineering Salmonella vaccineen_HK
dc.subject.meshAntigens - genetics - metabolism-
dc.subject.meshCloning, Molecular - methods-
dc.subject.meshGene Expression-
dc.subject.meshRecombination, Genetic-
dc.subject.meshSalmonella typhi - genetics - metabolism-
dc.titleA method to generate recombinant Salmonella typhi Ty21a strains expressing multiple heterologous genes using an improved recombineering strategyen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0175-7598&volume=91&issue=1&spage=177&epage=188&date=2011&atitle=A+method+to+generate+recombinant+Salmonella+typhi+Ty21a+strains+expressing+multiple+heterologous+genes+using+an+improved+recombineering+strategy-
dc.identifier.emailWatt, RM:rmwatt@hku.hken_HK
dc.identifier.emailZheng, BJ:bzheng@hkucc.hku.hken_HK
dc.identifier.emailYuen, KY:kyyuen@hkucc.hku.hken_HK
dc.identifier.emailHuang, JD:jdhuang@hkucc.hku.hken_HK
dc.identifier.authorityWatt, RM=rp00043en_HK
dc.identifier.authorityZheng, BJ=rp00353en_HK
dc.identifier.authorityYuen, KY=rp00366en_HK
dc.identifier.authorityHuang, JD=rp00451en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1007/s00253-011-3317-0en_HK
dc.identifier.pmid21611798en_HK
dc.identifier.scopuseid_2-s2.0-79959280769en_HK
dc.identifier.hkuros192276en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-79959280769&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume91en_HK
dc.identifier.issue1en_HK
dc.identifier.spage177en_HK
dc.identifier.epage188en_HK
dc.identifier.isiWOS:000291600600015-
dc.publisher.placeGermanyen_HK
dc.relation.projectToward the treatment of breast cancer by targeting breast tumor initiating cells with microRNAs-
dc.identifier.scopusauthoridYu, B=50263644200en_HK
dc.identifier.scopusauthoridYang, M=7404926247en_HK
dc.identifier.scopusauthoridWong, HYB=53364682700en_HK
dc.identifier.scopusauthoridWatt, RM=7102907536en_HK
dc.identifier.scopusauthoridSong, E=7101904256en_HK
dc.identifier.scopusauthoridZheng, BJ=7201780588en_HK
dc.identifier.scopusauthoridYuen, KY=36078079100en_HK
dc.identifier.scopusauthoridHuang, JD=8108660600en_HK
dc.identifier.citeulike9405864-

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