IBDV丝素蛋白/壳聚糖DNA微球疫苗的制备及免疫原性分析

Preparation and immunogenicity of silk fibroin/ chitosan microspheres for DNA vaccine delivery against infectious bursal disease virus

摘要:

为了制备传染性法氏囊病病毒 (Infectious bursal disease virus, IBDV) DNA微球疫苗，并评价其免疫效果。以丝素蛋白 (Silk fibroin, SF) /壳聚糖 (Chitosan, CS) 为壁材，IBDV的VP2/4/3 DNA疫苗为芯材，通过戊二醛和Na2SO4介导的乳化交联技术，制备出SF/CS复合微球疫苗，然后经肌肉注射14日龄非免疫鸡，2周后加强免疫一次，酶联免疫法 (ELISA) 定期监测鸡血清的IBDV抗体，以研究微球化疫苗的免疫原性。结果显示：戊二醛介导交联方法影响荷载DNA疫苗的活性，而Na2SO4介导的交联方法操作简单且不影响荷载DNA活性；建立了以壳聚糖浓度0.5% (pH 5.0)、丝素蛋白浓度0.6%，质粒DNA 500 μg/mL溶解在2% Na2SO4溶液中的工作条件；SF-CS复合微球DNA疫苗荷载率89.14%，大小1.98 μm，对DNaseⅠ的消化有保护作用。免疫后的抗IBDV血清ELISA抗体的检测显示，微球免疫组总体高于质粒疫苗免疫组(P<0.05)，而且SF/CS复合微球组免疫反应要略高于单纯CS包被的抗原组。研究表明，丝素蛋白/壳聚糖作为微球佐剂能提高IBDV DNA疫苗的临床免疫效果，有很好的应用前景。

Abstract:

To evaluate the immunities of biodegradable microsphere as a release delivery system for DNA vaccine against Infectious Bursal Disease Virus, in our study, silk fibroin/chitosan microsphere adjuvant was prepared with a precipitation/coacervation method. Both glutaraldehyde and Na2SO4 solution were used in cross-linking. No immune chicken were intramuscularly inoculated at 14 day-old and boosted 2 weeks later. The results show that glutaraldehyde destroyed the DNA activity of the vaccine whereas Na2SO4 solution did not. Factors of the chitosan concentration 0.5% (pH 5.0), silk fibroin concentration 0.6%, plasmid DNA (500 μg/mL) dissolved in 2% Na2SO4 solution were optimized to produce microsphere, with a loading capacity of 89.14%. The average particle size of SF-CS/pCI-VP2/4/3 microsphere is 1.98 μm, and it can protect the loading DNA vaccine from DNase I digestion. Data from anti IBDV ELISA antibodies in the serum show that immunization activity of the microsphere groups were generally higher than plasmid vaccine group (P<0.05), and the SF/CS compound microspheres group was better than that of sole CS microsphere group. The developed SF/CS microspheres are a very promising vaccine delivery system.