کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5801046 | 1555378 | 2013 | 8 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Toxoid construction of AsaP1, a lethal toxic aspzincin metalloendopeptidase of Aeromonas salmonicida subsp. achromogenes, and studies of its activity and processing Toxoid construction of AsaP1, a lethal toxic aspzincin metalloendopeptidase of Aeromonas salmonicida subsp. achromogenes, and studies of its activity and processing](/preview/png/5801046.png)
AsaP1 is a toxic aspzincin metalloendopeptidase secreted by the fish pathogen Aeromonas salmonicida subsp. achromogenes. The protease is highly immunogenic and antibodies against AsaP1 evoke a passive protection against infection with A. salmonicida subsp. achromogenes. The protease is expressed as 37Â kDa pre-pro-protein and processed to an active enzyme of 19Â kDa in A. salmonicida subsp. achromogenes. Recombinant expression of AsaP1rec in E. coli results in a protease of 22Â kDa that is not secreted. AsaP1rec induces comparable pathological changes in Atlantic salmon (Salmo salar L.) to native AsaP1wt. The aim of the study was to construct AsaP1 toxoids by exchanging catalytically important amino acids in the active site region of the protease.Four different AsaP1 mutants (AsaP1E294A, AsaP1E294Q, AsaP1Y309A, and AsaP1Y309F) were successfully constructed by one step site directed mutagenesis, expressed in E. coli BL21 C43 as pre-pro-proteins and purified by His-tag affinity chromatography and gel filtration. Three of the resulting mutants (AsaP1E294A, AsaP1E294Q, and AsaP1Y309A) were not caseinolytic active and are detected as unprocessed pre-pro-proteins of 37Â kDa. Caseinolytic active AsaP1rec and a mutant with reduced activity, AsaP1Y309F, were processed to a size of 22Â kDa. Furthermore, AsaP1rec is able to process the inactive mutants to the mature size of 22Â kDa, allowing the conclusion that AsaP1 is autocatalytically processed.All four mutants AsaP1E294A, AsaP1E294Q, AsaP1Y309A and AsaP1Y309F are non-toxic in fish but induce a specific anti-AsaP1 antibody response in Arctic charr (Salvelinus alpinus L.) and are therefore true toxoids and possible vaccine additives.
Journal: Veterinary Microbiology - Volume 162, Issues 2â4, 23 March 2013, Pages 687-694