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Streptococcus agalactiae (SA), which was often isolated from
bovine mammary gland, is one of the major pathogens causing
bovine mastitis. In order to study the optimum expression
conditions and the stability of surface protein "X" on SA, we at
inital step isolated it by employing hot-acid extraction method
followed with the FPLC purification. A protein with molecular
weight of ca. 100 kDa was then identified as "X" protein by a
SDS-PAGE analysis.Serum containing antibody against "X" protein
(Anti-"X" serum)was obtained by immunization of rabbit with the
purified "X" protein. By employing the anti-"X" serum, an ELISA
protocol capable of detecting the concentration of X protein on
the surface of SA was successfullly developed.Studies on the
expression of "X" protein with the developed ELISA protocol
revealed that :(1). different amounts of X protein was expressed
on various SA strains (i.e., X type, Ic/X type, and Ia/X type),
(2). SA grown under different conditions such as at 28℃, pH6,
shaking, and added with 5 % bovine serum expressed nearly the
same amount of "X" protein as under normal conditions (i.e.,
37℃, pH 6.7, semi-anaerobically), (3). a significant increase
of "X" protein was detected on SA grown under 5 % CO2
atmosphere, (4). SA grown under pH 8 or substitution of the
peptone ingredient in the Todd-Hewitt medium (TH medium) with
casein had expressed significantly less amount of X protein ,
(5). decline of X protein expression at the lag phase of SA
culture were also found on the growth conditions including
substitution of dextrose with lactose and addition of 5% whey in
the TH medium.With regard to the stability of "X" protein ,
following results were obtained : (1).conditions including (a)
storge at 4℃ for 32 days, and (b) repeated freeze and thaw
didn't cause the decline of "X" protein titer, (2) conditions
including storage at (a) -70℃for 120 days, (b)-20℃ for 60
days, (c) 25℃ for 16 days, (d) 37℃for 13 days, (e) 45℃ for
one day, and (f) 63℃ instantly were found to have a significant
decline effect on "X" protein titer, (3). SA culture treated
with trypsin also cause the loss of "X" protein titer."X" type
SA have been found to be a dominant serotype among bovine-origin
SA isolates. Recently, "X" protein was even identified as an
excellent target for opsonin and was capable of causing immune
response in cattle. Studies of optimum expression condition as
well asthe stability of "X" protein in this report may aid in
providing some information for the future development of bovine
mastitis vaccine.
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