空肠弯曲菌外膜蛋白亚单位疫苗的研制及免疫原性研究

Preparation of subunit vaccine with liposomal adjuvant for campylobacter jejuni and its immunogenicity

目的探索用脂质体包裹空肠弯曲菌（Camffylobaeter jejuni，CJ）Mr28000～31000外膜蛋白制备安全、稳定、有效的cJ亚单位脂质体佐剂疫苗及其免疫原性、免疫剂量和途径。方法采用薄膜一超声一冻融法制备阴离子脂质体（AL）、阳离子脂质体（CL）GJ疫苗后对其进行质量鉴定；用ELISA（间接法）检测经GJ疫苗免疫后的新西兰兔血清IgG抗体水平。结果①疫苗无菌试验及热原质试验均合格，均无明显不良反应；②阴、阳离子脂质体疫苗冻融前包裹率分别为32．1％、22．5％，冻融后分别是38．3％、28．4％；③CJ阴离子组比GJ阳离子组血清IgG的OD值显著增高（P〈0.01），与GJ弗氏组无统计学差别（P〉0.1）；CJ阴、阳离子组0．50mg／kg的免疫剂量血清IgG的0D值最大；CJ阳离子皮下注射组与肌肉注射组IgG的0D值无统计学差别（P〉0．1）；特异性IgG抗体经6个月动态观察，5个月后抗体效价下降。结论①采用薄膜-超声-冻融法制备GJ亚单位佐剂疫苗安全、稳定，冻融法可以提高脂质体疫苗包裹率；②同一剂量的阴离子脂质体疫苗血清抗体水平明显高于阳离子脂质体疫苗；③脂质体疫苗最佳接种剂量为0．50mg／kg，肌肉注射与皮下注射免疫效果无显著差异；④较高水平的特异性抗体维持5个月，6个月后应加强免疫。

Objective To explore the envelopment of the outer membrane protein （ Mr 28 000 - 31 000） with liposomes and prepare a safe, stable, and effective subunit vaccine of Campylobacterjejuni （CJ） and study on its immunogenicity. Methods The two liposomal subunit vaccines were prepared by the thin film diffusion-sonication-frozen melted method, and their quality were certificated. The specific IgG antibodies in serum of the New Zealand rabbits vaccinated with the CJ subunit vaccines were successively detected by indirect ELISA. Results （1） Pyrogen test, sterile test, and the side effects of the two vaccines were qualified. （2） Before frozen, the encapsulation ratio of anionic and cationic lipesomal vaccine wel＇e 32.1% and 22.5 %, respectively, but they were 38.3 % and 28.4% after frozen. （3） Specific IgG in serum of anionic lipesomal vaccine group was significant higher than that of cationic lipesomal vaccine group （ P 〈 0.01）, but similar to that of complete Freud＇ s adjuvant vaccine group （ P 〉 0.1 ）. In the 0.50 mg/kg dose, the IgG antibody levels in serum of anionic and cationic lipesomal vaccine group were both the highest. The IgG level in subcutaneous injection group had no difference from that of muscular injection group （ P 〉 0.1 ）. The level of specific IgG had been dynamically observed for six months and it dropped after five months. Conclusion （1） The thin film diffusionsonication-frozen melted method can be used to prepare the safe and stable subunit vaccine of CJ. Encapsulation ratio of the vaccines could be enhanced by frozen melted method. （2） In the same dose, the levels of IgG in serum of anionic lipesomal vaccine group are much higher than that of cationic lipesomal vaccine group. （3） The optimum dose of lipesomal vaccine is 0.50 mg/kg. Both subcutaneous injection and muscular injection can be used to vaccinate. （4） The high level of specific IgG can maintain five months, and the CJ vaccines should be strengthened after six months.