空肠弯曲菌外膜蛋白抗血清的制备及其对肠道病原菌鉴别的初步应用  

Preparation of Antiserum to outer membrance protein of Campylobacter jejuni and identification of its on enterobacteria

作　　者：冯胜军[1] 肖政[2] 孙万邦[2] 姚新生[2] 李国明[1] 刘仿[1] 陈群[1] 

机构地区：[1]广东医学院东莞校区微生物与免疫学教研室,523808 [2]贵州遵义医学院免疫学教研室,563003

出　　处：《医学信息（下旬刊）》2010年第6期10-11,13,共3页Medical information

基　　金：湛江市科技攻关项目（2006c03002）;广东医学院青年基金项目（XQ0410）

摘　　要：目的：探讨空肠弯曲菌（Campylobacter jejuni，CJ）28KD-31KD外膜蛋白兔抗血清的制备及其对常见肠道病原菌的检测的菌属特异性。方法：采用CJ活菌静脉注射、CJ28KD-31KD蛋白多次、多点皮下注射分别免疫新西兰种大白兔制备抗血清，Western—blotting法鉴定抗血清的抗原性。纯培养的痢疾志贺氏茸、甲型副伤寒沙门氏菌、肖氏沙门菌，希氏沙门菌、肠产毒性大肠埃希菌、变形杆菌、空肠弯曲茸（CF-1）（茼浓度1X109CFU／m1）等常见的肠道病原菌，分别与不同稀释倍数的CJ活菌抗血清和CJ28KD-31KD外膜蛋白抗血清进行常规的直接凝集反应（试管法），观察抗血清对各种已知肠遣病原菌的属的特异性。结果：制备了cJ28KD-31KD外膜蛋白的兔抗血清，CJ活菌抗血清能够与全部CJ甘氨酸提取物发生免疫反应，CJ28KD--31KD外膜蛋白抗血清仅与28KD-31KD外膜蛋白发生反应。CJ活菌抗血清能够与常见肠道病原体发生凝集反应，而CJ28KD-31KD蛋白抗血清仅与空肠弯曲菌（CF—1）发生凝集反应。结论：CJ28KD-31KD外膜蛋白兔抗血清被成功制备，该类蛋白或其中的某些重要组分可能是一种重要的CJ区别其他肠道病原菌的重要目标成分。该类蛋白成分的抗血清的成功制备，将为CJ的诊断血清建立，以及CJ亚单位、基因工程疫苗的研制提供初步的实验依据。Objective：To explore preparation for the antiserum for outer membrane protein with MWt of 28KD-31KD of Campylobaeter jejuni （antiserum to 28KD-31KD OMP） and identification group-specificity of AS of 28KD-31KD OMP to detect enteropathogen. Method, The outer membrane protein of Campylobaeter jejuni （OMP） was extracted by 0. 2mol/L.pH2.2 glyeine-HCL buffer solution and 28KD-31KD OMP of its was purificated by chromatographic fractionation with Sephadex G-75 and identified by SDS-PAGE; Rabbits were inoculated respectively via subcutaneous injection with 28KD-31KD OMP, or intravenous injection with viable organism of CJ,for up to three or more times. Two antiserums titers for CJ and 28KD-31KD OMP in serum from inoculated Rabbits were tested by double agar diffusion, and which were 1：16 or more. Two antiserum were used for testing antigenieity of OMP by Western-blotting. Group-specificity of pathogenic bacteria （1X109CFU/ml）, including of S. dysenteriae, S. paratyphi A, S. schottmuelleri, S. hirsehfeldii, enterotoxigenic E. coli, Bacillus proteus,CJ（strain CF- 1）, were identified with antiserum for OMP or 28KD-31KD OMP by the routine direct agglutination action （tubedilution）, respectively. Results： Rabbit antiserum to 28KD-31KD OMP was prepared（double agar diffusion test for dilution 1,16） successfully. Antiserum to CJ had immunoreaction with all OMP and all athogenie bacteria, but antiserum to 28KD-31KD OMP had immunoreaction with 28KD-31KD OMP of CJ and CJ only. Conclusion： Rabbit antiserum to 28KD-31KD OMP was prepared successfully,All or part of 28KD-31KD OMP were presumed the element for discrimination between Car and other athogenie bacteria. Rabbit antiserum to 28KD-31KD OMP would offer experimental and theoretical foundation for diagnostic serum establishment for CJ, and for CJ vaccine development.

关 键 词：空肠弯曲菌 外膜蛋白 抗血清 制备 诊断 

分 类 号：R446[医药卫生—诊断学]