| CpGODN佐剂序列的筛选及其免疫刺激活性研究 |
| 投稿时间：2015-09-18 修订日期：2015-11-02 点此下载全文 |
| 引用本文：赵玉娇,蔡路奎,王晓丹,席珏敏,陈俊英,潘玥,邱丽娟,姜黎明,孙强明.CpGODN佐剂序列的筛选及其免疫刺激活性研究[J].医学研究杂志,2016,45(5):37-42 |
| DOI：
10.11969/j.issn.1673-548X.2016.05.009 |
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| 基金项目:国家自然科学基金资助项目(面上项目)(81171946);教育部高校博士学科点专项科研基金资助项目(20111106120055);教育部留学回国人员启动基金资助项目[(2011)1568];云南省科技厅社会发展科技计划项目(2011CA016);云南省自然科学基金资助项目(2012FB188) |
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| 中文摘要:目的 设计和筛选对人PBMC细胞和小鼠脾细胞具有免疫刺激活性的CpGODN序列,研发能用于人用疫苗的有效核酸佐剂。方法 根据具有免疫刺激活性的CpGODN序列结构特点,设计并合成出15条候选序列,分别合成并进行非硫代和硫代修饰。分选健康人PBMC细胞及Balb/c小鼠脾细胞,以不同CpGODN进行体外刺激,MTT法检测CpGODN诱导细胞增殖水平。随后将筛选出的有效CpGODN序列与人类基因组序列进行同源性比较,以评估其安全性。结果 在刺激人PBMC和小鼠脾细胞72h后,硫代和非硫代修饰的15条序列A值均高于阴性对照组,其中IMB-AC5序列刺激人PBMC后A值分别达到0.377和0.334,与阳性对照ISS1018序列A值(0.387和0.338)接近;刺激小鼠脾细胞72h后,A值同样高于其他序列,达到0.571和0.486,与阳性对照ISS1018序列A值(0.588和0.464)接近。综合上述结果,选择5号非硫代序列作为候选佐剂。同源性比较结果显示,该序列与人类基因组序列没有同源性,与部分蛋白编码基因的同源性只有50%~65%,此外其部分序列与人18号染色体上的一些重复序列有同源性,最高同源性达62%。证明筛选出的新型CpGODN佐剂IMB-AC5安全性较高。结论 设计筛选出对人PBMC细胞和小鼠脾细胞具有免疫刺激活性的CpGODN序列IMB-AC5,与人类基因组序列具有较低同源性,为进一步研发人用疫苗佐剂奠定基础。 |
| 中文关键词:CpG ODN 佐剂 免疫刺激活性 同源性 |
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| Designing and Immunostimulation Activation Study of CpG ODN Adjuvants |
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| Abstract:Objective To design and screen out a CpG ODN sequence which has good immunostimulation activation on human PBMCs and mice spleen cells, to develop effective nucleic acid adjuvant for human vaccines. Methods According to the structure characteristics of CpG ODN sequences which have immunostimulation activities, 15 candidate sequences were designed and synthesised, with and without phosphorothioate modified respectively. Healthy PBMC cells and Balb/c mice spleen cells were Sorted out, and stimulated with different CpG ODN in vitro. CpG ODN inducing cells proliferation were detected by MTT method. Then for evaluating its safety, the homology of effective CpG ODN sequence were compared with human genome sequence. Results After 72h stimulating by CpG ODN in human PBMCs and in mice spleen cells, A values of totally 15 sequences (both phosphorothioate modified and non- modified) were higher than the negative control group, and A values of IMB- AC5 sequence in PBMCs was 0.377 and 0.334, close to the positive control ISS1018 sequence (0.387 and 0.338); and the A value in mice spleen cells reached to 0.571 and 0.486, close to the positive control ISS1018 sequence (0.588 and 0.464). Comprehensive the results above, the IMB-AC5 non-phosphorothioate modified sequence were chosen as the candidate adjuvant. The homology of its sequence was compared with the human genome sequence. The results showed that the sequence was not homogenous with human gene sequences, and the homology with part of protein-coding genes were between 50%-65% only, besides part of its sequence were homogenous with some repetitive sequences on chromosome 18. The highest homologies only reach to 62%, which showed that the new CpG ODN adjuvants IMB-AC5 had good saftey. Conclusion We designed and screened out an effective CpG ODN squence IMB- AC5, which has good immunostimulation activation on human PBMCs and mice spleen cells, and has low homology with the human genome sequence, lay a foundation for further development of adjuvants for human vaccines. |
| keywords:CpG ODN Adjuvant Immunostimulation activation Homology |
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