空肠弯曲菌28KD^31KD外膜蛋白的初步提取及鉴定

Extraction and identification of outer membrance protein with molecular mass of 28 KD ～ 31 KD of campylobacter jejuni

目的:探讨空肠弯曲菌28KD^31KD外膜蛋白的提取方法及其鉴定。方法:采用改良布氏肉汤培养基培养空ν淝?Campylobacterjejuni,CJ),用0 2mol/L、pH2 2甘氨酸-HCl缓冲液提取CJ外膜蛋白,SephadexG-75分子筛层析法纯化28KD^31KD分子,SDS-PAGE分子量鉴定,Westernblotting印迹法鉴定CJ甘氨酸提取物中外膜蛋白及纯化的28KD^31KD分子抗原性。结果:以甘氨酸提取CJ的外膜蛋白,SephadexG-75层析纯化能够提取出CJ28KD^31KD外膜蛋白,该蛋白分子能够与CJ兔抗血清发生特异性反应。结论:采用甘氨酸提取、SephadexG-75纯化CJ28KD^31KD外膜蛋白系一种稳定可靠的分离层析方法,提取的CJ28KD^31KD外膜蛋白具有良好的抗原性,为特异性的CJ28KD^31KD外膜蛋白的多克隆抗体制备、CJ感染的血清学特异性诊断、CJ亚单位疫苗的研制打下基础。

Objective:To explore extraction,purification and identification of outer membrane protein of Campylobacter jejuni (CJ).Methods: CJ(CF-1) was cultured on the improved Brucella broth medium, and extracted outer membrane protein of it by the 0.2 mol/L 、pH 2.2 glycine-hydrochloride buffered solution,and purified the outer membrane protein with molecular mass of 28 KD ～ 31 KD (P) by Sephadex G-75. P was identified with SDS-PAGE. and identified antigenicity of P with Western blotting by antiserum of rabbit immunized with live CJ. Results: The outer membrane protein with molecular mass of 28 KD ～ 31 KD of CJ could been extracted by 0.2 mol/L 、pH 2.2 glycine-Hydrochloride buffered solution ,and separated by Sephadex G-75 from the outer membrance protein, and had antigenicity. Conclusion:Sephadex G-75 was a available technique for extraction and purification the outer membrane protein with molecule mass of 28 KD ～ 31 KD of CJ that had antigenicity and will be worthy of preparation for its polyclone antiserum,and diagnosis for infection of CJ and research of subunit vaccine of CJ.[