肠炎沙门菌脂多糖抗原的制备及其生物学特性

Preparation and Identification of Salmonella enteritidis Lipopolysaccharide Antigen

目的制备肠炎沙门菌(Salmonella enteritidis,SE)脂多糖(Lipopolysaccharide,LPS)抗原,并鉴定其生物学特性。方法分别用热酚水法和水饱和冷酚法提取SE LPS抗原,免疫BALB/c小鼠,制备LPS单克隆抗体。采用蒽酮-硫酸法测定LPS多糖含量,考马斯亮蓝染色法测定其蛋白含量,SDS-PAGE分析其分子结构,Western blot分析其反应原性,间接ELISA法分析其特异性。结果热酚水法水相LPS粗提物中的多糖和蛋白含量分别约为3.413 mg/ml和46μg/ml,水饱和冷酚法水相LPS粗提物中的多糖和蛋白含量分别约为2.248 mg/ml和31μg/ml;提取的LPS呈现明显的梯度型条带;制备的2株抗LPS单抗(1D3和2E5)均能与提取的LPS发生特异性反应;制备的LPS除了与伤寒沙门菌A～F多价血清发生交叉反应外,与其他各种肠道菌单价或多价血清均未发生反应。结论热酚水法和水饱和冷酚法均可制备出活性、纯度均较好的LPS,但水饱和冷酚法操作简单,且提取的样品纯度高,特异性和反应原性良好,可用于肠炎沙门菌特异性抗原位点单抗的制备及免疫学检测。

Objective To prepare Salmonella enteritidis（SE） lipopolysaccharide（LPS） antigen and analyze its biological characteristics.Methods SE LPS antigen was extracted by hot phenol water and saturated cold phenol methods respectively and used for immunization of BALB/c mice to prepare monoclonal antibody（McAb）.The LPS was determined for polysaccharide content by anthrone-sulfuric acid method,for protein content by Coomassie brilliant blue staining,for molecular structure by SDS-PAGE,for reactogenicity by Western blot,and for specificity by indirect ELISA.Results The polysaccharide and protein contents in crude extracts of LPS prepared by hot phenol water method were 3.413 mg/ml and 46 μg/ml,while those by saturated cold phenol method were 2.248 mg/ml and 31 μg/ml,respectively.The prepared LPS showed obvious ladder bands on SDS-PAGE profile.Both prepared McAbs 1D3 and 2E5 showed specific reactions with the extracted LPS.The prepared LPS showed no reactions with any monovalent or polyvalent antisera against other enteric bacteria,except polyvalent antisera against SE types A ～ F.Conclusion The LPS with high activity and purity was prepared by both hot phenol water and saturated cold phenol methods.However,saturated cold phenol method was simpler to handle,by which the extracted sample showed higher purity,specificity and reactogenicity.The method may be used for preparation of specific McAb against SE LPS antigen and immunological determination of SE.