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

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 500μg/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1.98μm,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。

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 NaESO4 solution did not. Factors of the chitosan concentration 0.5% （pH 5.0）, silk fibroin concentration 0.6%, plasmid DNA （500 ~tg/mL） dissolved in 2% NaESO4 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.