不同培养基条件来源空肠弯曲菌外膜蛋白纯化及其表达差异  

Extraction,identification and express abundance of outer membrane protein of Campylobacter Jejuni cultured from different culture media

作　　者：冯胜军[1] 孙万邦[2] 肖政[2] 姚新生[2] 米娜[1] 刘仿[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,东莞523808 [2]遵义医学院免疫学教研室,563003

出　　处：《免疫学杂志》2010年第7期615-619,共5页Immunological Journal

基　　金：广东医学院青年基金项目(XQ0410);湛江市科技攻关项目(2006C03002)

摘　　要：目的探讨不同培养基培养条件下空肠弯曲菌(Campylobacter jejuni,CJ)Mr28000～31000外膜蛋白的纯化方案及表达差异。方法分别采用改良布氏血琼脂培养基(以下简称Bull’s)、改良卵黄培养基(以下简称Yolk)培养CJ,比较两者的生长特征。制备CJ全菌抗血清。0.2mol/L、pH2.2甘氨酸-HCl缓冲液抽取CJ外膜蛋白,Sephadex G-75层析法纯化其中的Mr28000～31000蛋白。SDS-PAGE技术检测Mr28000～31000外膜蛋白表达情况和含量差异,Western-blotting检测该类蛋白的抗原性。结果卵黄培养基培养的细菌生长更典型,SephadexG-75层析能够稳定可靠地纯化CJ细菌Mr28000～31000外膜蛋白,卵黄培养基培养来源的CJ的Mr28000～31000表达量占酸提取物的70%,大于改良布氏血琼脂培养基培养来源的表达量48%,两种培养基来源的CJ的Mr28000～31000蛋白成分均与CJ全菌兔抗血清发生免疫反应。结论不同培养基来源CJ的Mr28000～31000外膜蛋白均能被SphadexG-75分子筛纯化,该类蛋白能够在改良卵黄培养基上更高效表达,不同培养基来源的该类蛋白均具有良好的抗原性,该培养基的使用和该类蛋白有效提取为CJ感染的血清学特异性诊断及疫苗的研制奠定基础。This project aimed to extraction,purification,identification and express abundance of outer membrane protein（OMP） with relative molecular mass of 28 000-31 000 of Campylobacter jejuni（CJ） cultured via different culture media.We cultured CJ via the improved medium containing yolk,the improved Bull＇s blood agar,respectively.Then OMP of CJ extracted by the 0.2 mol/L glycine-hydrochloride buffered solution（pH2.2）,in which the OMP with relative molecular mass of 28 000-31 000（OMP 28 000-31 000） purified by chromatography with Sephadex G-75.Expressed abundance of OMP 28 000-31 000 was identified and compared by SDSPAGE,and antigenicity of OMP 28 000-31 000 was identified with Western-blotting by using antiserum of rabbit immunized with live CJ.CJ was grown better on the improved medium containing yolk.OMP of CJ from two culture media could be extracted by 0.2 mol/L glycine-Hydrochloride buffered solution（pH2.2）.OMP 28 000-31 000 with antigenicity could be separated by chromatography with Sephadex G-75.The OMP abundance of CJ cultured via the improved medium containing yolk is more high（70%） than that of the improved Bull＇s blood agar（48%）.We concluded that the improved medium containing yolk is a more productive medium for expression of the OMP 28 000-31 000 of CJ.And chromatography with Sephadex G-75 is a stable,efficient separation method for the OMP 28 000-31 000 of CJ.OMP 28 000-31 000 of CJ would be worthy of preparation for diagnosis reagent,and subunit vaccine of CJ.

关 键 词：空肠弯曲菌 培养基 外膜蛋白 SephadexG-75 纯化 

分 类 号：R392.11[医药卫生—免疫学]