肠出血性大肠杆菌O157:H7脂多糖抗原的制备及鉴定

THE PREPARATION OF LIPOPOLYSACCHARIDE ANTIGEN OF ENTEROHEMORRHAGIC ESCHERICHIA COLI O157: H7 AND ITS IDENTIFICATION

目的提取纯化肠出血性大肠杆菌（EHEC）O157：H7脂多糖（LPS）抗原并对其鉴定，以探讨其应用于检测抗O157抗体的价值。方法应用热酚水法提取EHECO157：H7S型933株LPS抗原，并经超速离心纯化。提取物以蒽酮硫酸法、紫外光谱吸收法、考马斯亮蓝染色法及鲎试剂分别测定多糖、核酸、蛋白含量和尝试剂凝集活性。LPS抗原致敏醛化鸡血球，IHA法检测EHEC157：H7及其它肠道病原菌的抗血清。对凝集阳性的抗血清，经菌体抗原吸收、O157LPS抑制及去脂质多糖抗原抑制试验，确定抗血清与LPS的反应位置。结果纯化后的O157LPS抗原告多精，具鲎试剂凝集活性，未检出核酸，蛋白含量＜0．5％。O157LPSIHA检测O157多克隆和单克隆抗体均呈高效价凝集阳性反应，而大部分其它肠道病原菌抗血清均呈阴性反应，交叉反应仅见于沙门氏菌O30、EIECO144：K？及ESIECⅣ抗血清。EHEC157和沙门氏菌O30抗血清与O157LPS反应位置为多糖部分，而EIECO144：K？及ESIECⅣ抗血清的反应位置为脂质A部分。结论热酚水法提取的EHECO157LPS抗原纯度高、特异性强，可望应用于抗O157抗体的检测。O157LPS抗原的去脂质可进一步提高反应的特异性。

Aim The lipopolysaccharide(LPS) antigen of enterohemorrhagic Escherichia coli(EHEC) O157: H7 wasprepared and identified to estirnate the value of its application in detecting for anti-EHEC O157 antibody. Methods The LPSantigen was prepared from EHEC O157: H7 strain 933 with hot phenol water method and purified by ultracentrifugation. Thecontents of polysaccharide,nucleic acid and protein in the extract and its activity of agglutinating limulus amoebcyte lysatewere detected. The antigen was used to detect EHEC O157: H7 and other enteric pathogenic bacteria antisera by IHA. Thereaction positions between LPS and the antisera having positive agglutinating reaction were identified by bacteria somatic anti-gen absorption test,Ol57 LPS antigen and polysaccharide antigen inhibition test. Results The O157 LPS antigen containspolysaccharide and has activity of agglutinating limulus amoebcyte lysate,in which nuc1eic acid has not been detected out andprotein content is less than O. 5 % (w/w). Anti - Ol57 polyclone and monoclone antibodies both react intensely to the O157LPS antigen. There are no cross-reaction between the antigen and most antisera of other enteric bacteria,while cross-reactionsare just found between the antigen and antisera of Salmonela O3O, EIEC O144 I K?,ESISC IV. EHEC Ol57 and SalmonelaO3o antisera react to polysaccharide of Ol57 LPS- EIEC Ol44 1 K? and ESIEC IV antisera react to lipid A of O157 LPS. Cou-clusiou The O157 LPS antigen has high purity and reaction specificity. lt could be used to detect anti-O15? antibody and re-action specificity may be raised further if lipid A is excluded from Ol57 LPS.