[Cloning and expression of hemolytic-toxin from Actinobacillus pleuropneumoniae and the immunoprotection in mice].
The ApxIIA, ApxIIIA, ApxIVA genes from Actinobacillus pleuropneumoniae serotype 3 and the ApxIA gene from Actinobacillus pleuropneumoniae serotype 5 were respectively cloned into the prokaryotic expression vector pGEX-5X-3. Then the recombinant expression plasmids were respectively transformed into E. coli BL 21 and fusion protein expression were induced by IPTG. The expression products were purified by precipitation with ammonium sulfate and chromatography on Sephadex G-200. SDS-PAGE indicated that the productsexpressed at a high level when the recombinant E. coli BL21 was induced 2h, joining IPTG to final concentration 1 mmol/L. Western blot analysis showed that the expression products had immunogenicity and specificity. Subunit vaccines were made by different purified expression products and Freund's adjuvant. Mice were immunized at 30 days and 45 days with the subunit vaccines. Then the mice were challenged with the APP of serotype 1, 3, 5, 7 or 10 at 60 days. The result of animal immunoprotection test showed that subunit vaccines (ApxIA + ApxIVA, ApxIA + ApxIIIA + ApxIVA, ApxIA + ApxIIA + ApxIIIA + ApxIVA) could offer 58.4%, 66.6%, 91.7% protection in mice against the challenge of serotype 1, 5 and 7 APP, respectively. These results suggested that the recombinant proteins had good immunogenicity and the subunit vaccine containing four kind of recombinant proteins could induce better immunoprotection.
Du
Aiqing
A
College of Animal Science and Technology, Shandong Agricultural University, Tai'an 271018, China. aiqingdu@163.com
Diao
Youxiang
Y
Zhang
Wei
W
Zhang
Ruiping
R
Zang
Dapeng
D
Liu
Fangna
F
D000189:Actinobacillus Infections / Q000276:immunology* / Q000517:prevention & control*; D016977:Actinobacillus pleuropneumoniae / Q000235:genetics / Q000276:immunology*; D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D001428:Bacterial Vaccines / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D008297:Male; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D011994:Recombinant Proteins