Instability of the major soluble antigen produced by Renibacterium salmoninarum

A DNA fragment containing all but the first 90 base pairs of g e n e msa encoding p57, the major soluble antig e n of Renibacterium salmoninarum, was cloned in the plasmid vector pUC18 and subsequently a soluble fusion protein was produced using the pMAL expression vector system. The fusion p r o t e~n retained major epitopes shared with the native p57 molecule and provided a source of protein suitable for further ~mmunologlcal analysis which was independent of in v~tro cultures of this slow growing organism. Antiserum raised a g a~n s t the purified fusion protein was used to probe Western blots of cell extracts and extracellular products denved from R. salmoninarum cultured in vitro. The results show that under cond~tions of iron-restr~ction, both the production and processing of p57 are reduced.

mentioning

confidence: 99%

mentioning

confidence: 99%

Production of the major soluble antigen of Renibacterium salmoninarum in Escherichia coli K12

Grayson¹,

Evenden²,

Gilpin³

et al. 1995

“…However, further studies would need to be conducted to discount the possibility of a comigrating protease. If such autolytic activity can be confirmed, the 72 kDa antigen may be similar to that the 57 kDa major antigen of Renibacterium salmoninarum, the aetiologic agent of bacterial kidney disease (Griffiths & Lynch 1991). Further characterisation of the autolytic behavior and substrate •: sequence deletion; -: conservation of an amino acid;…”

Section: Discussionmentioning

confidence: 98%

Characterisation of ISAV proteins from cell culture

Griffiths¹,

Cook²,

Mallory³

et al. 2001

The characterisation of 2 infectious salmon anemia virus (ISAV) proteins is described. Proteins were harvested from ISAV-infected Chinook salmon embryo (CHSE)-214 cell culture by continuous elution denaturing gel electrophoresis, enabling the harvest of specific molecular weight fractions. Through the use of a polyclonal antiserum to ISAV, it was possible to identify a potentially autolytic major antigen of 72 kDa and a glycosylated protein of approximately 38 kDa which varied in size depending on cell line compatibility. N-terminal amino acid sequencing of the glycosylated proteins suggests that it is encoded by segment 6 of the ISAV genome. Further, sequence analysis of the glycosylated protein account for the variable molecular weight and may explain differences in host cell compatibility. KEY WORDS: SAV · Proteins · Electrophoresis · GlycoproteinResale or republication not permitted without written consent of the publisher

“…This mechanism requires p57 to posess proteolytic activity. Lynch and collegues have demonstrated that p57, and several lower molecular weight products of p57, continue to degrade during 2-dimensional electrophoretic separation of ECP (Gnffiths & Lynch 1991, Barton et al 1997. Furthermore, p57, and degradation products of p57 isolated from the WC strain of Renibacterium salrnoninarum, comigrate with PMSF-sensitive proteolytic activity detected by gelatin substrate gels.…”

Section: Fraction (Ml)mentioning

confidence: 99%

Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum

Wiens

Chien²,

Winton³

et al. 1999

Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish. Biological activities of secreted p57 include agglutination of salrnonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4Cl1, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a nlonomer that retains salrnonid leucocyte agglutinat~ng activity. The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57. When combinations of the MAbs were used in an antigen capture ELISA. the epitopes recognized by the 3 MAbs were shown to be sterically separate. However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed. These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed. Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity. A model in which p57 is released from R. salmoninarum as a biologically active monomer during infection of salmonid fish is proposed.