阪崎肠杆菌脂多糖抗原的制备及其生物学特性

Preparation and biological characters of lipopolysaccharide antigen of Enteobacter sakazaii

目的采用不同方法提取并纯化阪崎肠杆菌(Enteobacter sakazaii,ES)脂多糖(Lipopolysaccharide,LPS)抗原,并对其生物学特性进行鉴定。方法常规培养ES,分别采用热酚水法和冷酚水法提取LPS抗原,收集水相和酚相LPS,采用蒽酮-硫酸法测定LPS多糖含量,紫外光谱法测定核酸含量,Bradford法测定蛋白含量,SDS-PAGE测定相对分子质量;以其免疫小鼠,间接ELISA法检测小鼠血清抗体效价,并检测其与各种肠道菌单价或多价血清的交叉反应性。结果 ES在相同条件下保存相同时间,采用冷酚水法提取LPS的产率高于热酚水法;所提取的水相和酚相LPS样品相对分子质量分布在14 400～45 000之间,均具有良好的免疫原性,且仅与ES单价血清反应,而与其他各种肠道菌单价或多价血清均不发生反应。结论热酚水法和冷酚水法均可制备免疫原性良好、特异性强的ES LPS抗原,但热酚水法所得样品的糖含量和蛋白含量均高于冷酚水法。

Objective To extract and purify lipopolysaccharide（LPS） antigen from Enteobacter sakazaii（ES） by various methods and identify its biological characters.Methods ES was cultured by routine method,from which LPS antigen was extracted by hot and cold phenol water methods respectively.The LPS in water and phenol phases was collected,and determined for polysaccharide content by anthrone-sulfuric acid method,for nucleic acid content by UV spectroscopy,for protein content by Bradford method,and for relative molecular mass by SDS-PAGE.Mice were immunized with the prepared LPS antigen,then determined for serum antibody titer by indirect ELISA,and for cross reactivity with monovalent or multivalent antisera against various intestinal bacteria.Results The yield of LPS extracted from ES stored at the same condition for the same time by cold phenol water method was higher than that by hot phenol water method.The relative molecular masses of LPS in water and phenol phases ranged from 14 400 to 45 000.Both the LPS antigens showed high immunogenicity and only reactions with monovalent antiserum against ES,while no reactions with monovalent or multivalent antisera against other intestinal bacteria.Conclusion Both hot and cold phenol water methods were suitable for preparation of immunogenic and specific LPS antigen of ES.However,both the polysaccharide and protein contents of antigens prepared by hot phenol water method were higher than those by cold phenol water method.