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

Chen, L. P. and Wu, D. F. and Cai, X. W. and Guo, F. J. and Blackall, P. J. and Xu, X. J. and Chen, H. C. (2012) Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector. Veterinary Microbiology, 155 (2-4). pp. 310-316. ISSN 0378-1135

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Article Link(s): http://dx.doi.org/10.1016/j.vetmic.2011.08.020

Abstract

The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glasser'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 x 10(2) CFUhlg of DNA. Transformation efficiency was notably increased (1.3 x 10(5) CFU/mu g 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. (C) 2011 Elsevier B.V. All rights reserved.

Item Type:Article
Business groups:Animal Science
Additional Information:ISI Document Delivery No.: 910HC Times Cited: 2 Cited Reference Count: 26 Chen, Liping Wu, Dongfang Cai, Xuwang Guo, Fengjuan Blackall, P. J. Xu, Xiaojuan Chen, Huanchun National Natural Science Foundation of China [30901074, 31072153]; National Basic Research Program (973) of China [2012CB518801]; Special Fund for Agro-scientific Research in the Public Interest [201003060] This work was supported by the National Natural Science Foundation of China (No. 30901074 and 31072153), the National Basic Research Program (973) of China (No. 2012CB518801) and the Special Fund for Agro-scientific Research in the Public Interest (201003060). We are grateful to Dr. Paul R. Langford (London, England) for the gift of plasmid pLOF/TAG and E. coli strain CC118 lambda pir. Elsevier science bv Amsterdam
Keywords:Haemophilus parasuis Shuttle vector Electransformation In vitro modification actinobacillus-pleuropneumoniae transformation restriction identification resistance genes expression efficiency barriers strains
Subjects:Animal culture > Swine
Veterinary medicine > Veterinary parasitology
Deposited On:10 Apr 2014 01:45
Last Modified:10 Apr 2014 01:45

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