Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector

Chen, Liping, Wu, Dongfang, Cai, Xuwang, Guo, Fengjuan, Blackall, P. J., Xu, Xiaojuan and Chen, Huanchun (2012) Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector. Veterinary Microbiology, 155 2-4: 310-316.


Author Chen, Liping
Wu, Dongfang
Cai, Xuwang
Guo, Fengjuan
Blackall, P. J.
Xu, Xiaojuan
Chen, Huanchun
Title Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector
Formatted title Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector
Journal name Veterinary Microbiology   Check publisher's open access policy
ISSN 0378-1135
1873-2542
Publication date 2012-03-23
Year available 2011
Sub-type Article (original research)
DOI 10.1016/j.vetmic.2011.08.020
Volume 155
Issue 2-4
Start page 310
End page 316
Total pages 7
Place of publication Amsterdam, Netherlands
Publisher Elsevier
Collection year 2012
Language eng
Formatted abstract The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glässer's disease in pigs, using a novel H. parasuis–Escherichia coli shuttle vector. A 4.2 kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800 bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a shuttle vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5 × 102 CFU/μg of DNA. Transformation efficiency was notably increased (1.3 × 105 CFU/μg of DNA) with vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel shuttle vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen.
Keyword Haemophilus parasuis
Shuttle vector
Electransformation
In vitro modification
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Available online 30 August 2011

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2012 Collection
Queensland Alliance for Agriculture and Food Innovation
 
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