壳聚糖/pcDNA-DPV-gC基因在雏鸭体内的抗原表达和分布

 【目的】以DPV gC基因疫苗（pcDNA-DPV-gC）为模型，采用复凝法制备壳聚糖/pcDNA-DPV-gC纳米微球，对其在雏鸭体内的抗原表达和分布进行初步研究。【方法】将壳聚糖/pcDNA-DPV-gC基因疫苗以肌注、滴鼻和口服3种途径免疫20日龄雏鸭，于免疫后不同时间点（4h、12h、1d、3d、5d、7d、2 w、4 w、6 w和10 w）分别随机宰杀2只雏鸭，采集肝、脾、肺、肾、胰、脑、胸腺、哈氏腺、法氏囊、食道、十二指肠、盲肠和直肠，应用间接免疫组化检测DPV gC基因在雏鸭体内的抗原表达和分布。【结果】①肌注组免疫雏鸭1 d，肝脏、法氏囊、十二指肠、盲肠和直肠检测到DPV gC蛋白；滴鼻组免疫雏鸭12 h，肺脏出现阳性信号，1 d哈氏腺和法氏囊检测到DPV gC蛋白；口服组免疫雏鸭12 h，食道出现中等强度的阳性信号，1 d法氏囊、十二指肠、盲肠和直肠检测到DPV gC蛋白；②在3种免疫途径中，肝脏、肺脏、法氏囊、哈氏腺、食道、十二指肠、盲肠和直肠是DPV gC抗原表达的主要器官；阳性信号主要在肝细胞、肺上皮细胞、法氏囊和哈氏腺淋巴细胞、食道上皮细胞和肠道粘膜上皮细胞及固有层细胞等部位；③不同免疫途径免疫的壳聚糖/pcDNA-DPV-gC基因疫苗在雏鸭体内的抗原表达量和持续时间的总体表达规律为：肌注组＞滴鼻组＞口服组。【结论】壳聚糖能促进DPV gC基因在雏鸭体内的表达和分布，肌肉注射是壳聚糖/pcDNA-DPV-gC基因疫苗首选的免疫方式。

Antigenic Expression and Distribution of Chitosan/pcDNADPV-gC Gene in the Vaccinated Ducklings

【Objective】 DNA-chitosan nanoparticles carrying duck plague virus (DPV) gC gene were constructed by using complex coacervation process to investigate its antigenic expression and distribution in the vaccinated ducklings. 【Method】 The 20-day-old DPV-free ducklings were respectively immunized with chitosan/pcDNA-DPV-gC gene vaccine via intramuscular injection, nasal administration and oral administration. At intervals of 4 h, 12 h, 1 d, 3 d, 5 d, 7 d, 2 w, 4 w, 6 w and 10 w post-vaccination (p.v.), two ducklings were randomly euthanatized and their organs (liver, spleen, lung, kidney, pancreas, brain, thymus, Harderian gland, bursa of Fabricius, esophagus, duodenum, caecum, and rectum) were collected. Meanwhile, an indirect immunohistochemical staining (IHC) was developed to detect antigenic expression and distribution of DPV gC antigens in the vaccinated ducklings. 【Result】 The DPV gC proteins were found in the liver, bursa of Fabricius, duodenum, caecum and rectum in the intramuscular injection group at 1 d post-vaccination (p.v.); Moreover, the immunogenicity were firstly found in the lung at 12 h p.v., and the DPV gC proteins were observed in the bursa of Fabricius and Harderian gland in the nasal administration group at 1 d p.v. Furthermore, the positive signals were firstly found in the esophagus at 12 h p.v., and the DPV gC proteins were observed in the bursa of Fabricius, duodenum, caecum and rectum in the oral administration group at 1 d p.v.. The DPV gC proteins were distributed in the liver, lung, bursa of Fabricius, Harderian gland, esophagus, duodenum, caecum and rectum, which were served as the principal sites for DPV gC antigens localization. The positive immunogenicity was mainly distributed in the parenchymal hepatic cells, epithelial cells of lung, lymphocytes of Harderian gland and bursa of Fabricius, epithelial cells of esophagus, epithelial cells and lamina propria mucosae cells of intestinal tract. According to the immunogenicity intensity and duration time via different immunization routes, the positive staining in all tissues was in the order of intramuscular injection group＞nasal administration group＞oral administration group. 【Conclusion】 The results demonstrated that chitosan as a polycationic gene carrier could promote the expression efficiency of DPV gC antigens in the vaccinated ducklings. Intramuscular injection was considered to be the best immunization routes to inoculated chitosan/pcDNA- DPV-gC gene vaccine.