不同剂量鸭瘟病毒gC基因疫苗在雏鸭体内的表达时相和分布规律

Antigenic Distribution and Expression Regularity in the Different Doses of pcDNA-DPV-gC DNA Vaccine in Tianfu Ducklings

本研究利用已建立的间接免疫组化方法检测鸭瘟病毒(DPV)gC基因疫苗(pcDNA-DPV-gC)免疫雏鸭后其表达蛋白在雏鸭体内的表达时相和分布规律,为DPV gC基因疫苗的进一步研究和应用提供基础数据。将不同剂量(50、100和200μg)DPV gC基因疫苗免疫3周龄天府肉鸭,于免疫后不同时间点(4h、12h、1d、3d、5d、7d、2周、4周、6周和10周)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、十二指肠、盲肠、直肠以及注射部位肌肉,应用间接免疫组化方法检测pcDNA-DPV-gC在雏鸭体内的表达时相和分布规律。结果显示:①各剂量免疫组第1天在肝、十二指肠、盲肠、直肠和注射部位肌肉检测到DPV gC蛋白,其中注射部位肌肉的阳性信号最强;第2周时各组织中的抗原表达量达到高峰,随着时间推移,阳性信号以不同速度逐渐衰减;第10周时各剂量免疫组仍在肝、脑、十二指肠、盲肠和直肠发现持续表达DPV gC蛋白;而胰在所有时间点均未检出阳性信号;②pcDNA-DPV-gC在不同组织的表达量也存在差异,肝、脾、法氏囊、脑、十二指肠、盲肠和直肠是DPV gC蛋白主要的分布器官;阳性信号主要出现在肠黏膜固有层细胞、脾白髓或红髓区的淋巴细胞以及脑皮质神经胶质细胞等部位;③根据各组织的阳性信号强度和持续时间的情况,得到pcDNA-DPV-gC在雏鸭各组织的总体表达规律:十二指肠、盲肠、直肠>肝脏>法氏囊>脾>脑>注射部位肌肉>胸腺>肺>哈氏腺>肾脏>胰脏;④不同剂量pcDNA-DPV-gC免疫雏鸭后各组织中抗原表达量和持续时间的总体规律依次为200μg组>100μg组>50μg组。结果表明不同剂量pcDNA-DPV-gC免疫后1d即可在雏鸭体内发现阳性信号,持续存在10周仍可检测到DPV gC蛋白,预示pcDNA-DPV-gC免疫期可持续较长时间。

The objective of the present study was to develop and apply an indirect immunohistochemistry method for detecting the antigenic distribution and expression regularity of pcDNA-DPV-gC in the experimentally vaccinated duckling tissues. Three-week-old Tianfu ducking were inoculated with different doses （50, 100 and 200 μg） of DPV gC DNA vaccine by intramuscular injection. At intervals of 4 h, 12 h, 1 day, 3 days, 5 days, 7 days, 2 weeks, 4 weeks, 6 weeks, and 10 weeks post-vaccination （p.v.）, two ducklings were randomly euthanatized and their organs （liver, spleen, lung, kidney, pancreas, brain, thymus, Harderian gland, bursa of Fabricius, duodenum, caecum, rectum and muscle of injected spot） were collected. Meanwhile, the indirect immunohistochemistry method was established to detect the antigenic distribution and expression regularity of pcDNA-DPV-gC in the experimentally vaccinated ducking tissues. The results showed that：①The positive staining were found in the liver, duodenum, caecum, rectum and muscle of injected spot at 1 day p.v., and the immunoreactivity in the muscle of injected spot was stronger than other organs. In addition, a drastic reduction of DPV gC antigen level were observed after the highest immunoreactivity at 2 weeks p.v., but still had a detectable vaccine antigen level in the liver, brain, duodenum, caecum and rectum at 10 weeks p.v.. Interestingly, there was scarcely detected positive signal in the pancreas throughout the immunologic process; ②The positive staining reaction for pcDNA-DPV-gC had a conspicuous discrepancy among the different tissues. The positive immunogenicity was mainly found in the liver, spleen, bursa of Fabricius, brain, duodenum, caecum, and rectum, which served as the principal sites for antigen localization. The DPV gC encoding glycoprotein could be expressed in the various types of cells, especially in the lamina propria mucosae cells of intestinal tract, the lymphocyte of splenic white pulp and red pulp and the nerve cement cells of pallium. ③According to the immunogenicity intensity and duration time, the positive staining in all tissues were in below order： duodenum, caecum, rectum liver bursa of Fabricius spleen brain muscle of injected spot thymus lung Harderian gland kidney pancreas. ④The signal intensity and duration time of different doses were in below order： 200 μg group 100 μg group 50 μg group. Different doses of pcDNA-DPV-gC could continuously express DPV gC encoding glycoprotein as early as 1 day p.v., and exist in those at least 10 weeks p.v. The results demonstrated that the immunization time of pcDNA-DPV-gC was a long period located in the experimentally vaccinated ducking tissues.