基因枪轰击不同剂量小鹅瘟病毒VP3基因疫苗在雏鹅体内的动态分布

Dynamic Distribution of GPV-VP3 Gene Vaccine in Geese Vaccinated with Gene-gun Bombardment

本文开展了检测小鹅瘟病毒(GPV)VP3基因的实时荧光定量PCR(FQ-PCR)方法的建立和基因枪轰击不同剂量(1、3和6μg)GPV-VP3基因疫苗(pcDNA-GPV-VP3)在30日龄四川白鹅体内(心、肝、脾、肺、肾、法氏囊、胸腺、哈氏腺、十二指肠、空肠、回肠、直肠、盲肠、胰腺、血液、脑及注射部位皮肤)分布规律的研究。结果表明:①建立的FQ-PCR特异性强、灵敏度高、重复性好,核酸模板数与FQ-PCR测定的Ct值相关系数达到0.999,具有很好的直线相关性;②pcDNA-GPV-VP3各剂量免疫雏鹅1 h即可在各组织中检测到,其中注射部位含量最高,肝、肾、淋巴器官(脾、法氏囊、胸腺、哈氏腺)含量较高;③到免疫后217 d时,1μg组免疫雏鹅各个组织器官内仍检测到pcDNA-GPV-VP3的存在,但多数组织器官中的含量比1 h时约少了4个数量级,其中免疫部位减少了7个数量级;④血液中pcDNA-GPV-VP3的含量较少,且免疫后1 h^217 d各时间点的差异不显著(P≥0.05);⑤不同剂量pcDNA-GPV-VP3免疫雏鹅各组织中的含量呈现的总体规律为6μg组>3μg组>1μg组,但差异不显著(P≥0.05)。因此,FQ-PCR是定量检测pcDNA-GPV-VP3在免疫雏鹅体内含量的可靠方法,pcDNA-GPV-VP3免疫雏鹅后1 h时可分布至雏鹅体内各组织器官中并持续存在217 d以上。

This paper concentrates on developing the Real-time PCR method for detecting GPV- VP3 gene and studying the dynamic distribution of GPV-VP3 gene vaccine （pcDNA-GPV-VP3）in geese （duodenum, liver, spleen, kidney, bursa of Fabricius, thymus, ileum, cecum, pancreas, heart, blood, jejunum, lung, gland of Harder, skin of injected spot, rectum and brain）. Ninety 30-day-old geese were divided into 5 groups. Animals in pcDNA-GPV-VP3 inoculation groups were injected via gene-gun with different doses（1, 3, 6 μg）, respectively. Results showed that. （1）This assay was specific, highly sensitive and rapid for detecting pcDNA-GPV-VP3. The standard curve showed a good linear relationship between Ct and template concentrations, and the correlation coefficient was 0. 999. （2）The pcDNA-GPV-VP3 could be detected in all tissues and peripheral blood 1 h post immunization. The copy numbers were most at the skin of injected spots, then liver, kidney, lymphoid organ （spleen, bursa of Fabricius, thymus, gland of Harder）. （3） After 217 d post-administration, pcDNA-GPV-VP3 could still be detected in tissues of 1 μg dose group. But the copy numbers decreased about 10^4 copies in the most tissues than that at 1 h post administration, the obvious decrease of pcDNA GPV-VP3 was found at the injection sites （it dropped about 107 copies ）. （4）The copy numbers in the serum were always little and changed not obvious with the time （ P≥0.05 ）. （5）Different dosage groups of pcDNA-GPV-VP3 distributed in the geese tissues did not significantly differ from each other（P≥0. 05）, the 6 μg group had the highest copy numbers, then the 3 μg group, and the 1 μg group had the lowest. The results demonstrated that FQPCR was a reliable method for detecting dynamic distribution of pcDNA-GPV- VP3, and pcDNA GPV-VP3 could distribute in all tissues of geese 1 h post immunization, and exist in those at least 217 days post immunization.