生物工程學(xué)報 Chin J Biotech 2008, March 25; 24(3): 363-367
journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061
[email protected] . 2008 Institute of Microbiology, CAS & CSM, All rights reserved
研究報告
豬產(chǎn)腸毒素大腸桿菌987P 蛋白受體的鑒定
朱國強1, 王建業(yè)1, 朱曉芳2
1 揚州大學(xué)獸醫(yī)學(xué)院, 揚州 225009
2 江蘇省蘇北人民醫(yī)院, 揚州 225001
摘 要: 產(chǎn)腸毒素大腸桿菌(ETEC)定植于仔豬腸道的第一步是通過987P 菌毛與小腸上皮細(xì)胞表面刷狀緣大分子(BBV)結(jié)合,。對分離的BBV 進行SDS-PAGE 和Ligand blot 分析表明, 在32~35KDa 區(qū)域內(nèi)有一條帶能被987P 菌毛探針?biāo)R別和結(jié)合, 所結(jié)合的條帶經(jīng)胰蛋白酶消化后, 通過微內(nèi)徑反相高效液相色譜(RP-HPLC)分離出多條主要峰帶蛋白峰帶,采用襯質(zhì)輔助激光解吸與電離質(zhì)譜法(MALDI-MS)對主要峰帶進行分析, 結(jié)合多肽氨基酸測序和Blast 同源性比較, 得到3 個氨基酸基序(AETAP、ALAAAGYDVEK 和LGLK), 其序列與人和鼠源的組蛋白H1 高度同源; 來源于仔豬小腸上皮細(xì)胞BBV 的H1 蛋白與BBV 一樣都能特異性結(jié)合純化的987P 菌毛蛋白,。上述結(jié)果表明, 仔豬小腸上皮細(xì)胞BBV 的組蛋白H1 是987P 菌毛蛋白的受體,。
關(guān)鍵詞: 產(chǎn)腸毒素大腸桿菌, 987P, BBV, 組蛋白H1, 受體
Identification of the 987P Protein Receptors for Enterotoxigenic Escherichia coli
Guoqiang Zhu1, Jianye Wang1, and Xiaofang Zhu2
1 College of Veterinary Medicine, Yangzhou University, Yangzhou 2250091, China
2 Northern Jiangsu People Hospital, Yangzhou 225001, China
Abstract: The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediates adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by Ligand blot, protein bands with a set of 32~35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC(RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequences of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The above results indicated that the 987P protein receptors are piglet BBV-derived Histone H1 proteins.
Keywords: enterotoxigenic Escherichia coli, 987P, BBV, Histone H1 proteins, receptor
在國內(nèi), 987P 產(chǎn)腸毒素大腸桿菌(ETEC)所致仔豬黃痢的嚴(yán)重性及多種疾病(如豬瘟、豬繁殖障礙綜合征等)與ETEC 混合,、并發(fā)感染的復(fù)雜性一直困擾著養(yǎng)豬業(yè)[1,2],。ETEC 與共生非致病性大腸桿菌不同,其致病性決定于它們在宿主上皮細(xì)胞上的定居能力和產(chǎn)生腸毒素的能力, 兩者缺一不可。ETEC 定居在小腸上皮細(xì)胞的能力是由菌體表面的宿主特異性腸吸附菌毛(粘附素)介導(dǎo)的,。病原菌感染機體的第一步也是最重要的一步就是粘附素和宿主細(xì)胞表面相應(yīng)的特異性受體結(jié)合, 而介導(dǎo)細(xì)菌的定居,、宿主細(xì)胞信號傳遞和適當(dāng)途徑的毒素傳遞, 從而引發(fā)機體的一系列的病理過程, 直至細(xì)胞、組織器官死亡,。因此, 從分子水平上研究病原菌粘附素與宿主細(xì)胞表面受體相互作用的精確機制, 才能最直接,、最有效地阻止病原菌侵入機體細(xì)胞, 這是當(dāng)前病原菌預(yù)防和控制研究的最活躍領(lǐng)域[7]。大多數(shù)菌毛能作為凝集素(lectin)介導(dǎo)與不同種類動物紅細(xì)胞相互作用,結(jié)合于宿主細(xì)胞表面糖蛋白或糖脂受體的糖基部位上,。與大多數(shù)ETEC 菌毛不同, 987P 不能凝集哺乳動物紅細(xì)胞或需用化學(xué)法處理紅細(xì)胞后才能獲得凝集性[8], 它僅能與易感仔豬小腸上皮細(xì)胞或腸上皮細(xì)胞刷狀緣大分子(BBV)結(jié)合, 但小腸上皮細(xì)胞BBV 大分子上存在何種蛋白質(zhì)(受體)與987P 粘附素結(jié)合？多年來獸醫(yī)界和生物學(xué)界試圖尋找987P 蛋白的受體而未果,。本文對987P 蛋白質(zhì)受體的分離和鑒定方法作如下研究,。
Received: May 16, 2007; Accepted: August 8, 2007
Supported by: National Natural Science Foundation of China (No. 30571374), Jiangsu National Science Foundation (No. BK2005049), the
projects-sponsored by SRF for ROCS, SEM and USDA project (No. 2002-35204-12216).
Corresponding author: Guoqiang Zhu. +86-514-87972590; Fax: 86-514-87972218; E-mail: [email protected], [email protected]
國家自然科學(xué)基金(No. 30571374); 江蘇省自然科學(xué)基金(No. BK2005049); 教育部留學(xué)回國人員科研啟動基金資助項目。部分工作在美國賓夕
法尼亞大學(xué)獸醫(yī)學(xué)院微生物實驗室完成和美國農(nóng)業(yè)部USDA 高新技術(shù)2002-35204-12216 部分資助,。
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