Cloning and expression in Escherichia coli of a protective antigen of Erysipelothrix rhusiopathiae

Sakakura

et al. 1999

We purified the protein antigen (P64), which contains 66 and 64 kDa proteins, from the alkaline extract (AE) of whole cells of Erysipelothrix rhusiopathiae strain Agata (serovar 5) to determine the protective activity of the antigen against E. rhusiopathiae infection in pigs. The serum titre of antibody against P64 rapidly increased in pigs immunized with 500 and 100 mg of P64 and reached maximum values at 3 weeks after the first immunization (1 week after the second immunization). However, the serum antibody titres were not increased in pigs immunized with 20 mg of P64 and in nonimmunized pigs. In the pigs immunized with live cell vaccine (acriflavin-fast attenuated strain Koganei 65-0.15), the serum titres of antibody against P64 also increased at 1-2 weeks after immunization. In a pig challenge test performed on immunized and nonimmunized pigs, all nonimmunized pigs showed typical clinical signs of swine erysipelas (fever, erysipeloid, arthritis), while all pigs immunized with 500 and 100 mg of P64 and live cell vaccine showed no clinical signs of this disease. In Western blot analysis, sera from pigs immunized with P64 and live cell vaccine strongly reacted with the 64 kDa protein. In contrast, the serum from nonimmunized pigs did not react with any proteins. From these results, it was suggested that a specific antibody against the 64 kDa protein could be increased in pigs immunized with P64 or live cell vaccine and that this anti-P64 antibody has a strong protective effect against E. rhusiopathiae infection in pigs. U. S.

Section: Western Blottingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

Protective Activity of the Purified Protein Antigen of Erysipelothrix rhusiopathiae in Pigs

Yamazaki

Sakakura

et al. 1999

“…The anti-CF antiserum conferred crossprotection of mice when they were challenged with serovars 1a, 1b, 2, 5, 6, 8, 11, 12, 15, 16, 18, 21 and N strain (SAWADA and TAKAHASHI, 1987;SAWADA et al, 1987c). GALÁ TIMONEY (1990) and reported that 64 and 66 kDa proteins have strong protective activity against E. rhusiopathiae infection in mice. In our previous studies (KOBAYASHI et al, 1992;SATO et al, 1995), we found that the CF and the AE of E. rhusiopathiae strain Agata protected mice against the infection caused by several serotypes of E. rhusiopathiae and antisera involved in protective activity against E. rhusiopathiae infection in mice recognized the 64, 66 and 43 kDa proteins in both the CF and the AE.…”

Section: Western Blotting Using Anti-p64 and Anti-p43 Serummentioning

confidence: 99%

“…However, the yield of protective protein antigen from the CF and the AE of E. rhusiopathiae and the degree of protective activities of P64 and P43 in a mouse protection assay were not determined. GALÁ N and TIMONEY (1990) reported that structural proteins of 66, 64 and 43 kDa were important for protection against E. rhusiopathiae infection in mice. also reported that a 64-66 kDa protein exhibited a high degree of protective activity in a mouse challenge test.…”

Section: Introductionmentioning

confidence: 99%

Isolation and Purification of a Protective Protein Antigen of Erysipelothrix rhusiopathiae

Miyazaki

Sakakura

et al. 1999

The rapid growth and high survival rate of Erysipelothrix rhusiopathiae was determined using a culture of the bacterium in tryptic soy broth supplemented with 0.3% Tris-hydroxymethyl aminomethane and 0.1% Tween 80 (TT-TS broth). High concentrations of 64, 66 and 43 kDa proteins, which are associated with protection against E. rhusiopathiae infection in mice, were obtained by alkaline treatment of whole cells using 0.05-1 N NaOH. The supernatant of alkaline treated cells (alkaline extract; AE) was stable at alkaline or neutral pH. However, aggregates appeared at neutral pH in the absence of sodium dodecyl sulphate (SDS). A high yield of 64, 66 and 43 kDa proteins was obtained from strain Agata (serovar 5). The proteins were eluted from gel bands following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the AE from strain Agata and designated P64 and P43. The amounts of P64 and P43 isolated were 0.7 and 0.3 mg/16 g of wet bacteria, respectively. In a mouse protection test, 50% protective doses (PD 50 ) of P64 and P43 were 0.58 and 0.63 mg, respectively. Upon Western blotting of the AE, both anti-P64 and anti-P43 antibodies reacted with the 64 and 43 kDa proteins. From these results, it is suggested that P64 is the most effective protective antigen and that P43 (43 kDa protein) is a degradation product of P64. Therefore, the 64 kDa structural proteins are associated with the induction of a protective activity against E. rhusiopathiae infection in mice.

“…GALÁ N and TIMONEY (1990) reported that the 66, 64 and 43 kDa bacterial structural proteins are important for the protection of mice against E. rhusiopathiae infection. GROSCHUP et al (1991) reported that the 66-64 kDa protein exhibited a high degree of protective activity in a mouse challenge test.…”

Section: Introductionmentioning

confidence: 99%

Preparation and Partial Characterization of Monoclonal Antibodies Against the Protective Protein Antigen of Erysipelothrix rhusiopathiae

Yamazaki

Kodaira

et al. 1999

Thirteen mouse monoclonal antibodies (Mabs) against the protective protein antigen (P64) of Erysipelothrix rhusiopathiae were prepared and partially characterized. The titres of the Mabs varied from 200 to 1 638 400 as determined by enzyme‐linked immunosorbent assay (ELISA). Of the 13 Mabs 10, two and one belonged to the IgG2a, IgG1 and IgM subclasses, respectively. All Mabs reacted strongly with the 64 kDa protein and weakly with the 43 kDa protein upon Western blotting of the alkaline extract (AE) of E. rhusiopathiae. The protective activity (pd50/ml) of the 13 Mabs against E. rhusiopathiae infection in mice varied from <50 to >50 000. These Mabs were classified into three groups, highly protective Mabs, moderately protective Mabs and Mabs which did not possess protective activity, based on the protective index (ratio of the pd50/ml to the antibody titre). These results suggest that the 64 kDa protein is an effective protective antigen, which is easily cleaved into many small proteins, including the 43 kDa protein, and possesses at least two epitopes related to its protective activity and at least one epitope which is not related to protection of mice against E. rhusiopathiae infection.