Jiangsu Agri-animal Husbandry Vocational College, Veterinary Bio-Pharmaceutical, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
Poult Sci. 2019 Jan 1;98(1):112-118. doi: 10.3382/ps/pey325.
The avian adeno-associated virus (AAAV) has been proved to be an efficient gene transfer vector for human gene therapy and vaccine research. In this experiment, an AAAV-based vaccine was evaluated for the development of a vaccine against duck hepatitis a virus type 1 (DHAV-1). The major capsid VP1 gene was amplified and subcloned into pFBGFP containing the inverted terminal repeats of AAAV, and then the recombinant baculovirus rBac-VP1 was generated. The recombinant AAAV expressing the VP1 protein (rAAAV-VP1) was produced by co-infecting Sf9 cells with rBac-VP1 and the other 2 baculoviruses containing AAAV functional genes and structural genes respectively, and confirmed by electron microscopy, Western blotting and immunofluorescence assays. Quantitative real-time PCR revealed that the titer of rAAAV-VP1 was about 9 × 1012 VG/mL. Immunogenicity was studied in ducklings. One day ducklings were injected intramuscularly once with rAAAV-VP1. Serum from rAAAV-VP1-vaccinated ducklings showed a systemic immune response evidenced by VP1-specific enzyme-linked immunosorbent assay and virus neutralization test. Furthermore, all ducklings inoculated with rAAAV-VP1 were protected against DHAV-1 challenge. The data of quantitative real-time RT-PCR from livers of challenged ducklings also showed that the level of virus copies in rAAAV-VP1 group was significantly lower than that of the PBS group. Collectively, these results demonstrate that the AAAV-based vaccine is a potential vaccine candidate for the control of duck viral hepatitis.
禽腺相关病毒（AAAV）已被证明是人类基因治疗和疫苗研究中有效的基因转移载体。在本实验中，评估了基于 AAAV 的疫苗在开发针对鸭甲型肝炎病毒 1 型（DHAV-1）的疫苗中的作用。扩增了主要衣壳 VP1 基因，并亚克隆到含有 AAAV 反向末端重复序列的 pFBGFP 中，然后生成重组杆状病毒 rBac-VP1。通过共感染 Sf9 细胞与 rBac-VP1 和另外两种分别含有 AAAV 功能基因和结构基因的杆状病毒，产生表达 VP1 蛋白的重组 AAAV（rAAAV-VP1），并通过电子显微镜、Western blot 和免疫荧光检测进行验证。实时定量 PCR 显示 rAAAV-VP1 的滴度约为 9×1012 VG/mL。在雏鸭中研究了免疫原性。雏鸭在 1 日龄时肌肉注射一次 rAAAV-VP1。rAAAV-VP1 免疫的雏鸭血清通过 VP1 特异性酶联免疫吸附试验和病毒中和试验显示出系统免疫反应。此外，所有接种 rAAAV-VP1 的雏鸭均对 DHAV-1 攻击具有保护作用。来自攻毒雏鸭肝脏的实时定量 RT-PCR 数据还表明，rAAAV-VP1 组的病毒拷贝数水平明显低于 PBS 组。综上所述，这些结果表明基于 AAAV 的疫苗是控制鸭病毒性肝炎的潜在疫苗候选物。
