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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。
Objective To investigate the purification scheme and expression difference of outer membrane proteins of Campylobacter jejuni (CJ) Mr28000 ~ 31000 under different culture conditions. Methods CJ was cultured with modified Brinell’s blood agar medium (hereinafter referred to as Bull’s) and improved yolk medium (hereinafter referred to as Yolk), respectively. Preparation of CJ whole bacterial antiserum. CJ outer membrane protein was extracted by 0.2 mol / L glycine-HCl buffer at pH2.2 and Mr28000 ~ 31000 protein was purified by Sephadex G-75 chromatography. SDS-PAGE technique was used to detect the expression and content of Mr28000 ~ 31000 outer membrane proteins, Western blotting was used to detect the antigenicity of these proteins. Results The growth of bacteria in yolk culture medium was more typical. SephadexG-75 chromatography could stably and reliably purify the outer membrane protein Mr28000 ~ 31000 of CJ bacteria. The expression level of Mr28000 ~ 31000 in culture medium of yolk culture medium accounted for 70% , Which was greater than 48% of the expression level in the modified Brucella blood agar medium. The protein components of Mr28000 ~ 31000 in CJ of the two culture mediums all immunoreacted with the CJ whole rabbit antiserum. Conclusion The outer membrane proteins of Mr28000 ~ 31000 from different culture media can be purified by SphadexG-75 molecular sieve. These proteins can be expressed more efficiently in modified yolk medium. The proteins of different culture media have good antigenicity , The use of this culture medium and the effective extraction of this kind of protein provide the basis for the serological diagnosis and vaccine development of CJ infection.