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| Construction of a Composite Attenuated Mutant Actinobacillus pleuropneumoniae Strain WF83ΔapxⅡC/apxⅠAN/apxⅠAC |
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Abstract Porcine contagious pleuropneumonia (PCP), caused by Actinobacillus pleuropneumoniae (APP), is an infectious porcine (Sus scrofa) respiratory tract disease causing severe economic losses worldwide in the swine industry. Aiming at developing a safe attenuated vaccine against multiple serotypes, the APP serotype 7 isolates WF83 strain (APP-7-WF83) based recombinant transfer vector pBSLNKAR was constructed, which contained the kanamycin resistance gene (Kan) as a resisitance screening gene replacing complete toxin Ⅱ activate gene C (apxⅡC), with the N-terminal and C-terminal fragment squence of toxinⅠstructural gene A (apxⅠA) of Actinobacillus pleuropneumoniae RTX-toxin (Apx) gene family from APP serotype Ⅰ isolates shope4074 strain inserted. The vector pBSLNKAR was electroporated into the parent strain APP-7-WF83 to build mutant strain WF83ΔapxⅡC/apxⅠAN/apxⅠAC. The antibodies expressed by N-terminal fragment squence of toxin Ⅰstructural gene A (apxⅠAN) neutralized the toxinⅠso that the mutant had no hemolysis compared with APP-7-WF83 by hemolytic detection. PCR identification showed that WF83ΔapxⅡC/apxⅠAN/apxⅠAC lacked about 377 bp of apxⅡC and was inserted about 377 bp of apxⅠAC and 1 188 bp of apxⅠAN compared with APP-7-WF83. With 0.5 mL of trypticase soy broth (TSB) (blank control), the parent strain APP-7-WF83 group and the mutant WF83ΔapxⅡC/apxⅠAN/apxⅠAC group inoculated mice (Mus musculus) with 1.1×109 cfu/mL, respectively. The results showed that the mice of parent strain groups died of 100%, the mice of mutant group had no death. The results confirmed that the gene deletion composite attenuated strain WF83ΔapxⅡC/apxⅠAN/apxⅠAC was successfully constructed, which supplies basic data for further research on gene deleted attenuated vaccine.
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Received: 26 November 2014
Published: 13 May 2015
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| [1]Frey J. Virulence in Actinobacillus pleuropneumoniae and RTX toxins [J]. Trends Microbial, 1993, 139(8):1723-8.[2]Haesebrouck F, Chiers K, Van Overbeke I, et al. Actinobacillus pleuropneumoniae infections in pigs: the role of virulence factors in pathogenesis and protection [J]. Vet Microbial, 1997,58(2-4):239-49.[3]Pattison IH, Howell DG, Elliot J. A haemophilus-like organism isolated from pig lung and the associated pneumonic lesions [J]. J Comp Pothole, 1957, 67(4):320-30.[4]Tascon R I, Vazquez-Boland J A, Gutierrez-Martin C B, et al. The RTX haemolysins ApxI and ApxII are major virulence factors of the swine pathogen Actinobacillus pleuropneumoniae: evidence from mutational analysis [J]. Mole Microbial, 1994, 14(2):207-16.[5]Van de Kerkhof A, Haesebrouck F, Chiers K, et al. Influence of Actinobacillus pleuropneumoniae and its metabolites on porcine alveolar epithelial cells [J]. Infect Immune, 1996, 64(9):3905-7. |
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