中国实用口腔科杂志

中国实用口腔科杂志

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牙龈卟啉单胞菌和具核梭杆菌多克隆抗体的制备及应用效果评价

张智颖a，刘东娟b，潘亚萍a   

  1. 中国医科大学附属口腔医院a牙周科，b综合急诊黏膜病科，辽宁  沈阳  110002
  • 出版日期:2021-05-30 发布日期:2021-06-03
  • 基金资助:
    国家自然科学基金（81870771，81800975）；中国博士后科学基金（2018M640269，2019T120224）

  • Online:2021-05-30 Published:2021-06-03

摘要： 目的    制备高效价高纯度的牙龈卟啉单胞菌（P. gingivalis）和具核梭杆菌（F. nucleatum）多克隆抗体，并评价其应用效果。方法    将新鲜培养的P. gingivalis和F. nucleatum菌液灭活后与佐剂乳化混匀，作为抗原皮下多点注射免疫雄性新西兰大白兔，定期免疫并在抗体效价达到预期后采用颈动脉取血获得抗血清。采用间接ELISA法测定多克隆抗体交叉反应。应用硫酸铵沉淀法纯化多克隆抗体，并通过Western-blot方法鉴定抗体的纯度。荧光显微镜观察多克隆抗体应用于免疫荧光实验的效果。结果    免疫6周后，血清抗体效价达到1∶320 000 ~ 1∶640 000，相互之间未出现交叉反应，且纯度较高。免疫荧光实验中可观察到明显的荧光，并可见P. gingivalis的球杆状形态和F. nucleatum的短杆状形态。结论    成功制备了P. gingivalis和F. nucleatum多克隆抗体，其特异性良好，为后续的免疫学相关实验奠定了基础。

关键词: 牙周炎, 牙龈卟啉单胞菌, 具核梭杆菌, 多克隆抗体

Abstract: Objective    To prepare high-titer and high-purity polyclonal antibodies against Porphyromonas gingivalis（P. gingivalis）and Fusobacterium nucleatum（F. nucleatum）and evaluate their application effect. Methods    The freshly cultured P. gingivalis and F. nucleatum strains were inactivated and then emulsified and mixed with adjuvants，and were used as antigen subcutaneous injections to immunize male New Zealand white rabbits. The rabbits were regularly immunized until the antibody titer reached expectation，and then the blood from carotid artery were taken to obtain antiserum. The indirect ELISA method was used to determine the cross-reaction of polyclonal antibody. The polyclonal antibody was purified by ammonium sulfate precipitation method，and the purity of the antibody was identified by Western-blot method. The fluorescence microscope was used to observe the effect of polyclonal antibody in immunofluorescence experiments. Results    The titer of the prepared serum antibody reached 1∶320 000 - 1∶640 000，there was no cross-reaction between each other，and the purity was high. Obvious fluorescence could be observed in the immunofluorescence experiment，and the club-shaped morphology of P. gingivalis and the short rod-shaped morphology of F. nucleatum could be seen. Conclusion    The polyclonal antibodies of P. gingivalis and F. nucleatum are successfully prepared with good specificity，which lays the foundation for subsequent immunology-related experiments.

Key words: periodontitis, Porphyromonas gingivalis, Fusobacterium nucleatum, polyclonal antibody