請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/13656
標題: 無乳練球菌表面抗原“X”蛋白的穩定度及表現性研究
Studies on the stability and expression characteristics of surface protein "X" on Streptococcus agalactiae
作者: 楊振杉
Yang, Chen Shan
關鍵字: 無乳鏈球菌
乳房炎
穩定度
表現性
X蛋白
Streptococcus agalactiae
mastitis
X protein
stability
expression
出版社: 獸醫學系
摘要: 中文摘要 無乳鏈球菌(Streptococcus agalactiae)是引起牛乳房炎的 主要病原菌,常可自感染乳腺的牛乳分離出。X蛋白是SA菌血清型中的 一個表面抗原,我們利用熱鹽酸法與FPLC法分離出X蛋白,得知X蛋白的 分子量約為100 kDa。 藉由抗X蛋白血清,以 ELISA法測試X蛋白的 表現性與穩定性。在X蛋白的表現性方面: (1)供試的X型、Ic/X型、 Ia/X型三個菌株,X蛋白的表現量各有差異;(2)細菌在28℃、pH 6、震 盪培養與加5%胎牛血清等環境下培養,X蛋白的表現不受影響;(3)在5 %CO2環境下,細菌X蛋白的表現顯著增加;(4)在pH 8、以 casein取代 peptone培養時,X蛋白表現極顯著減少;(5)以lactose取代dextrose、 加5%whey培養時,X蛋白在細菌生長初期減少。 在X蛋白的穩定性方 面:(1)SA菌置於-70℃,經過150天,X蛋白仍保持穩定;(2)置於-20℃ 時,第150天X蛋白開始下降;(3)置於 4℃經過32天仍保持穩定;(4)置 於25℃時第16天開始下降;(5)置於37℃時第13天開始下降;(6)置於45℃ 時第一天即下降;(7)置於63℃時瞬時即刻下降;(8)細菌經十次的反覆冰 凍解凍,X蛋白仍保持穩定;(9)細菌以trypsin處理時,部份的X蛋白會 被破壞。 在引起乳房炎的SA菌分離株中,X型菌株佔有較高的比例。 由於X蛋白是調理素的標的物,可引起免疫反應。經本實驗對X蛋白的表 現性與穩定性的研究,將有助於對X蛋白的瞭解,及對乳牛乳房炎的防治 。
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.
URI: http://hdl.handle.net/11455/13656
顯示於類別:獸醫學系所

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