碳纳米管载草鱼呼肠孤病毒DNA疫苗系统靶向免疫机制研究

Vaccine delivery technology for carbon nanotubes is one of the most effective means to make bath immunization has the same immune effect as injection immunization. Targeting applications for fish vaccines are feasible methods for improving the immune effects. On the basis of non-injectable carbon nanotubes grass carp revirus DNA vaccine system which has been prepared in our laboratory, the targeting carbon nanotubes DNA vaccine system which can recognize the signature receptors (mannose and Fc receptors) of immune cells will be constructed. The Fc receptor targeting recognition factor CH3 will be added into grass carp reovirus DNA vaccine by the use of genetic engineering technology. Meanwhile, this vaccine will be also modified by nectar glycated chitosan to improve the targeting of mannose receptor with chemical modification. At last, the carbon nanotubes DNA vaccine system which has dual targeting recognition factors will be constructed. The law of sustained release for vaccine system will be revealed by using fluorescence quantitative techniques. The pattern of vaccine delivery in fish will be measured by flow cytometry and immunofluorescence techniques. The immune mechanism will be measured by enzyme-linked immunosorbent assay and quantitative PCR technology. Protective efficacy of vaccine system will be measured by viral infection. The results in this study will be used to lay the foundation of carbon nanotube vaccine system achieve effective immune protection on fish, and also play an important role in supporting development of healthy aquaculture and ensuring the safety of aquatic products and ecology.

碳纳米管载疫苗技术的研究是解决大规模浸浴免疫达到注射免疫效果的有效手段之一，而渔用疫苗靶向性应用又是提高免疫效果切实可行的方法。本项目在前期构建免注射型碳纳米管载草鱼呼肠孤病毒DNA疫苗系统的基础上，提出构建以抗原呈递细胞表面标志性受体（甘露糖和Fc受体）为靶点的靶向性碳纳米管载疫苗系统，用于提高疫苗免疫效率和效果。通过基因工程和化学合成技术，选用抗体CH3和甘露糖化壳聚糖为靶向识别因子，构建双重靶向性碳纳米管载DNA疫苗系统；采用荧光定量技术等揭示疫苗系统的缓释作用规律；利用流式细胞术、免疫荧光定量技术等明确疫苗系统在体内的靶向呈递途径；通过酶联免疫吸附法和定量PCR技术等阐明其提高识别效率的作用机制；结合病毒攻毒验证其免疫保护效果。该项研究结果不仅为靶向性碳纳米管载DNA疫苗的应用提供了理论依据和研究模板，而且也对保障水产养殖健康发展、水产品和生态安全具有重要的意义。

碳纳米管载疫苗技术的研究是解决大规模浸浴免疫达到注射免疫效果的有效手段之一，而渔用疫苗靶向性应用又是提高免疫效果切实可行的方法。本项目在前期构建免注射型碳纳米管载草鱼呼肠孤病毒DNA疫苗系统的基础上，选用抗体CH3和甘露糖化壳聚糖为靶向识别因子，构建以抗原呈递细胞表面标志性受体（甘露糖和Fc受体）为靶点的双重靶向性碳纳米管载DNA疫苗系统，并验证其免疫保护效果。在此基础上，构建碳纳米管载甘露糖化VP7亚单位疫苗（CNTs-M-VP7），探究其缓释作用及靶向呈递规律，评价其免疫保护效果。结果显示：碳纳米管载DNA疫苗系统中疫苗承载率最高可达30%，与对照组和其他处理组相比（pEGFP-VP7、pEGFP-VP7-CH3、甘露糖化壳聚糖-DNA疫苗），碳纳米管载DNA疫苗系统组的血清抗体效价、免疫生理生化指标、免疫相关基因表达水平均显著提高。病毒攻毒后，碳纳米管载DNA疫苗系统的免疫保护率显著高于其他处理组。碳纳米管载甘露糖化VP7亚单位疫苗中甘露糖含量为4.3%，VP7抗原蛋白含量为48.34%。草鱼巨噬细胞、脾脏和肾脏组织对CNTs-M-VP7的摄取量显著高于其他处理组（VP7、CNTs-VP7）。活体成像分析显示免疫6 h后，草鱼对CNTs-M-VP7的摄取逐渐增加，肾脏和脾脏对CNTs-M-VP7的摄取随着时间的延长逐渐增加。与对照组和其他处理组相比（VP7、CNTs-VP7），CNTs-M-VP7组的血清抗体效价、免疫生理生化指标、免疫相关基因表达水平均显著提高。病毒攻毒后，VP7处理组和CNTs-VP7处理组的免疫保护率分别为58%和72%，而CNTs-M-VP7组的免疫保护率为96%，显著高于其他处理组。该项研究结果不仅为靶向性碳纳米管载DNA疫苗和亚单位疫苗提供理论依据和研究模板，而且对保障水产养殖健康发展和水产品安全、生态安全具有重要的意义。