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Cloning, expression and immunogenic characterization of the apxIIA gene of Actinobacillus pleuropneumoniae

Published online by Cambridge University Press:  15 June 2007

Yan Ke-Xia ,
Liu Jian-Jie ,
Wu Bin ,
Tang Xi-Biao ,
Cai Li-Jun ,
Yang Ming-Liu ,
Chen Huan-Chun  and
Zhou Rui
Yan Ke-Xia
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Liu Jian-Jie
Affiliation:
Haikou Agriculture, Industry & Trade Luoniushan Co. Ltd.Haikou 570125, China
Wu Bin
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Tang Xi-Biao
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Cai Li-Jun
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Yang Ming-Liu
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Chen Huan-Chun
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Zhou Rui*
Affiliation:
Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
*
*Corresponding author. E-mail: rzhou@mail.hzau.edu.cn
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Abstract

The structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA of Actinobacillus pleuropneumoniae (APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that the apxIIA gene was expressed in Escherichia coli BL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108 cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

Keywords

Actinobacillus pleuropneumoniaeApxIIcloningprokaryotic expressionimmunogenicity
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 4 , Issue 1 , April 2007 , pp. 63 - 67
Copyright
Copyright © China Agricultural University and Cambridge University Press 2007

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Footnotes

First published in Journal of Agricultural Biotechnology 2006, 14(4): 493–497

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