[Cloning and expression of the Apx IVA gene of Actionbacillus pleuroneumoniae and development of an indirect ApxIVA-ELISA].
Apx IV, a forth RTX toxin indentified in Actionbacillus pleuropneumoniae recently, is expressed by all A. pleuropneumoniae regardless the serotypes and inducible only in vivo toxin, so it is the optimal to develop species-specific and differentiated diagnostic assay. Here the 2445bp DNA fragment of apxIVA gene of A. pleuroneumoniae was amplified and fused in-frame to the downstream of the T7 promoter and 6 His Tag of the prokaryotic expression vector pET-28b. The construct was transformed into E. coli BL21(DE3). After induction by 1.0 mol/L IPTG, a recombinant protein about 90 kD in size, designed as ApxIVAN, was detected, which was present as inclusion bodies and reacted specifically with swine antisera to the APP-serotype-1 by dot-blot. An indirect ELISA (ApxIVA-ELISA) was developed using purified recombinant ApxIVAN from the inclusion bodies as described previously, which had excellent specificity to A. pleuroneunoniae. Using the ApxIVA-ELISA, the ApxIV antibodies were not detected in the inactivated APP bacterins vaccinated pigs, but were detected in A. pleuropneumoniae serotype 1, 2 and 7 infected pigs and mice. These results suggested that ApxIVA-ELISA can be used not only to detect all serotypes of APP, but also to differentiate the naturally infected and inactivated vaccine immunized pigs.
Huang
Hong-Liang
HL
Laboratory of Animal Virology, Huazhong Agaricultural University, Wuhan 430070, China.
Zhou
Rui
R
Chen
Mei-Ling
ML
Liu
Jian-Jie
JJ
Xu
Xiao-Juan
XJ
Chen
Huang-Chun
HC
D000189:Actinobacillus Infections / Q000175:diagnosis* / Q000382:microbiology / Q000662:veterinary; D016977:Actinobacillus pleuropneumoniae / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D015870:Gene Expression; D005798:Genes, Bacterial; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
C122177:ApxIVA protein, Actinobacillus pleuropneumoniae; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins