小鹅瘟病毒VP_3基因疫苗在免疫雏鹅体内动态分布及表达规律的研究
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摘要
本文围绕小鹅瘟病毒(GPV)VP3基因检测方法、GPV VP3基因疫苗及其GPV弱毒疫苗免疫雏鹅以后在机体分布及表达等开展了下列研究：①检测小鹅瘟病毒(GPV)VP3基因的实时荧光定量PCR(FQ-PCR)的建立，②将GPV-VP3基因疫苗(pcDNA-GPV-VP3)分别以每只200μg、100μg、50μg肌肉注射，6μg、-3μg、1μg基因枪轰击的方法免疫30日龄四川白鹅，然后用FQ-PCR和免疫组织化学的方法检测在动物体内(心、肝、脾、肺、肾、法氏囊、胸腺、哈氏腺、十二肠、空肠、回肠、直肠、盲肠、胰腺、血液、脑及注射部位皮肤)分布及表达规律，③将GPV弱毒疫苗以每只200μL肌肉注射免疫免疫30日龄四川白鹅做对照，比较其和GPV-VP3基因疫苗进入机体后分布及表达的差异。获得如下结果：
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。
2．pcDNA-GPV-VP3在雏鹅体内的分布规律：各剂量免疫雏鹅1h即可在各组织中被检测到，其中免疫部位含量最高，其次是肝、肾、淋巴器官(脾、法氏囊、胸腺、哈氏腺)；到217d时，各组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1h时约低了少了10~3-10~4，其中免疫部位减少了约10~4-10~5；血液中pcDNA-GPV-VP3的含量较少，1h比其他组织低10~1-10~2；随后含量开始下降，随着时间的推移，pcDNA-GPV-VP3在各组织器官中含量的下降速度有变慢的趋势，3d-21d约下降了10~(0.1)-10~(0.5)，到63d-217d含量仅下降了10~(0.5)-10~1；脑1h-12h含量呈上升趋势，12h时达到最大值，随后下降。
3．pcDNA-GPV-VP3在雏鹅体内的表达规律：pcDNA-GPV-VP3的表达产物1d时能在免疫部位中的细胞质中检测到，基因枪组1d阳性颗粒最多，肌肉组时达到最多，63d肌肉组免疫部位中仍能检测到阳性颗粒，肠腺和肠绒毛上的杯状细胞细胞质／心肌细胞核也能检测到较多阳性；肝细胞间质中有零星表达，肺、肾、脾、胰腺、脑、法氏囊、胸腺中未见表达。
4．不同途径免疫pcDNA-GPV-VP3后在雏鹅体内的的分布和表达差异：不同途径免疫后，pcDNA-GPV-VP3在雏鹅体内广泛分布，并在心、肝、肠道和免疫部位表达，在免疫早期，基因枪组中各组织中pcDNA-GPV-VP3的含量普遍大于肌肉注射组，心、肝中棕黄色阳性颗粒出现比肌肉注射组早，但到217d时两种免疫方式都未能检测到表达产物。
5．不同剂量与分布和表达的关系：不同剂量pcDNA-GPV-VP3免疫雏鹅各组织中的含量和表达量呈现的总体规律为6μg组＞3μg组＞1μg组，200μg组＞100μg组＞50μg组，说明基因疫苗的剂量和分布、表达呈现一定的正相关性；
6．GPV弱毒疫苗在雏鹅体侵染和排泄：GPV弱毒疫苗免疫雏鹅后，病毒含量1h-3d呈现下降后回升的趋势，7d时达到最大值，35d逐渐下降，1d-3d时能在免疫部位、心、肠道和肝中检测到病毒抗原，第7d量达到最大；与基因疫苗组相比，GPV弱毒疫苗在各组织内检测到的含量大，差异显著(0.01≤P≤0.05)，表达抗原数量多，但数量下降的很快，63d时，各组织内未能检测到抗原物质。
This study concentrates on①developing the Real-time PCR method for GPV-VP3 detection,②studing the dynamic distribution of GPV-VP3 gene vaccine (pcDNA-GPV-VP3)in geese (duodenum, liver, spleen, kidney, fabricius, thymus, ileum, gland of Harder, position of injected spot, rectum, cecum, pancreas, heart, blood, jejunum, lung, and brain), through 30-day-old geese were injected pcDNA-GPV-VP3 with different doses via gene-gun (6, 3, 1μg), intramuscular injection (200μg, 100μg, 50μg) respectively and live attenuated vaccine of GPV(200μL),③studing the dynamic distribution of live attenuated vaccine of Sichuan isolate of GPV.
Results showed that:
1. This assay was specific, highly sensitive, and rapid for detecting pcDNA-GPV-VP3. The standard curve showed a fine linear relationship between Ct and template concentration, and the correlation coefficient was 0.999.
2. Dynamic distribution of GPV-VP3 gene vaccine in gosling: The pcDNA-GPV-VP3 could be detected in all tissues and peripheral blood at 1h post infection, the copy number was most at the position of injected spot, then liver, kidney, lymphatic organ (spleen, fabricius, thymus, gland of Harder); At 217d post administration, pcDNA-GPV-VP3 could still be detected in tissues of different doses group. But the copy number decreased about 10~3-10~4 copies in the most tissues than it at 1h post administration, the obvious decrease was found at the injection sites (it dropped about 10~4-10~5 copies); The changement of the copy numbers in the serum was always little and at 1h post administration was less about 10~4-10~5 copies than other tissues; then the copy numbers decreased, with the time passed slowly, decreased 10~(0.1)-10~(0.5) in 3d-21d, only 10~(0.5)-10~1 in 63d-217d; in brain the copy number increased in 1h-12h, reach the peak at 12h, then deceased.
3. Dynamic expression of GPV-VP3 gene vaccine in gosling: The expression of pcDNA-GPV-VP3 could be detected at 1d post infection from the cytoplasm of injected spot, the positive numbe reached peak at 1d via vaccined with gene gun bombardment, and at 7d with intramuscular injection. At 63d post administration, expression could still be detected in the sarcoplasmand of injected spot, it also could be detected easier in cellular of cardiac muscle, caliciform cell in villi intertinales and gl. Intersinales, intercellular substance of liver; other tissues had no expression.
4. Different dynamic distribution and expression of GPV-VP3 gene vaccine in gosling via the different Immunogenicity: pcDNA-GPV-VP3 DNA vaccine immunized geese respectively by the different routes of gene gun bombardment and intramuscular injection, which all could be detected in all tissues, and expressed VP3 in liver, ileum, cecum, heart, jejunum, duodenum, rectum, position of injected spot; pcDNA-GPV-VP3 DNA immunized by gene gun bombard- ment demonstrated higher copy numbers in all tissues and earlier expressed in liver, cecum than intramuscular injection at nonage; but at 217d post administration were no different.
5. Different dynamic distribution and expression of GPV-VP3 gene vaccine in gosling with the different doses: The quantity of pcDNA-GPV-VP3 and expression detected in the experimental groups showed the relations with the quantity of the inoculation doses, but not proportion (P≥0.05).
6. Infection and excretion of attenuated vaccine of GPV in gosling: Geese were immunized with live attenuated vaccineof GPV, the copy number decreased at the beginning then increased in 1h-3d, the maximum amount at 7d post administration, after 35d the copy number decreased gradually; At the position of injected spot, heart, liver and intestinal, viral antigens were detected in 1d-3d, the maximum amount at 7d; compared with the DNA vaccine, attenuated GPV vaccine was detected more copy number and expression in tissues, the difference was significant (P≤0.01≤0.05); but the number dropped quickly, all tissues failed to be detected antigen at 63d.
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。
2．pcDNA-GPV-VP3在雏鹅体内的分布规律：各剂量免疫雏鹅1h即可在各组织中被检测到，其中免疫部位含量最高，其次是肝、肾、淋巴器官(脾、法氏囊、胸腺、哈氏腺)；到217d时，各组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1h时约低了少了10~3-10~4，其中免疫部位减少了约10~4-10~5；血液中pcDNA-GPV-VP3的含量较少，1h比其他组织低10~1-10~2；随后含量开始下降，随着时间的推移，pcDNA-GPV-VP3在各组织器官中含量的下降速度有变慢的趋势，3d-21d约下降了10~(0.1)-10~(0.5)，到63d-217d含量仅下降了10~(0.5)-10~1；脑1h-12h含量呈上升趋势，12h时达到最大值，随后下降。
3．pcDNA-GPV-VP3在雏鹅体内的表达规律：pcDNA-GPV-VP3的表达产物1d时能在免疫部位中的细胞质中检测到，基因枪组1d阳性颗粒最多，肌肉组时达到最多，63d肌肉组免疫部位中仍能检测到阳性颗粒，肠腺和肠绒毛上的杯状细胞细胞质／心肌细胞核也能检测到较多阳性；肝细胞间质中有零星表达，肺、肾、脾、胰腺、脑、法氏囊、胸腺中未见表达。
4．不同途径免疫pcDNA-GPV-VP3后在雏鹅体内的的分布和表达差异：不同途径免疫后，pcDNA-GPV-VP3在雏鹅体内广泛分布，并在心、肝、肠道和免疫部位表达，在免疫早期，基因枪组中各组织中pcDNA-GPV-VP3的含量普遍大于肌肉注射组，心、肝中棕黄色阳性颗粒出现比肌肉注射组早，但到217d时两种免疫方式都未能检测到表达产物。
5．不同剂量与分布和表达的关系：不同剂量pcDNA-GPV-VP3免疫雏鹅各组织中的含量和表达量呈现的总体规律为6μg组＞3μg组＞1μg组，200μg组＞100μg组＞50μg组，说明基因疫苗的剂量和分布、表达呈现一定的正相关性；
6．GPV弱毒疫苗在雏鹅体侵染和排泄：GPV弱毒疫苗免疫雏鹅后，病毒含量1h-3d呈现下降后回升的趋势，7d时达到最大值，35d逐渐下降，1d-3d时能在免疫部位、心、肠道和肝中检测到病毒抗原，第7d量达到最大；与基因疫苗组相比，GPV弱毒疫苗在各组织内检测到的含量大，差异显著(0.01≤P≤0.05)，表达抗原数量多，但数量下降的很快，63d时，各组织内未能检测到抗原物质。
This study concentrates on①developing the Real-time PCR method for GPV-VP3 detection,②studing the dynamic distribution of GPV-VP3 gene vaccine (pcDNA-GPV-VP3)in geese (duodenum, liver, spleen, kidney, fabricius, thymus, ileum, gland of Harder, position of injected spot, rectum, cecum, pancreas, heart, blood, jejunum, lung, and brain), through 30-day-old geese were injected pcDNA-GPV-VP3 with different doses via gene-gun (6, 3, 1μg), intramuscular injection (200μg, 100μg, 50μg) respectively and live attenuated vaccine of GPV(200μL),③studing the dynamic distribution of live attenuated vaccine of Sichuan isolate of GPV.
Results showed that:
1. This assay was specific, highly sensitive, and rapid for detecting pcDNA-GPV-VP3. The standard curve showed a fine linear relationship between Ct and template concentration, and the correlation coefficient was 0.999.
2. Dynamic distribution of GPV-VP3 gene vaccine in gosling: The pcDNA-GPV-VP3 could be detected in all tissues and peripheral blood at 1h post infection, the copy number was most at the position of injected spot, then liver, kidney, lymphatic organ (spleen, fabricius, thymus, gland of Harder); At 217d post administration, pcDNA-GPV-VP3 could still be detected in tissues of different doses group. But the copy number decreased about 10~3-10~4 copies in the most tissues than it at 1h post administration, the obvious decrease was found at the injection sites (it dropped about 10~4-10~5 copies); The changement of the copy numbers in the serum was always little and at 1h post administration was less about 10~4-10~5 copies than other tissues; then the copy numbers decreased, with the time passed slowly, decreased 10~(0.1)-10~(0.5) in 3d-21d, only 10~(0.5)-10~1 in 63d-217d; in brain the copy number increased in 1h-12h, reach the peak at 12h, then deceased.
3. Dynamic expression of GPV-VP3 gene vaccine in gosling: The expression of pcDNA-GPV-VP3 could be detected at 1d post infection from the cytoplasm of injected spot, the positive numbe reached peak at 1d via vaccined with gene gun bombardment, and at 7d with intramuscular injection. At 63d post administration, expression could still be detected in the sarcoplasmand of injected spot, it also could be detected easier in cellular of cardiac muscle, caliciform cell in villi intertinales and gl. Intersinales, intercellular substance of liver; other tissues had no expression.
4. Different dynamic distribution and expression of GPV-VP3 gene vaccine in gosling via the different Immunogenicity: pcDNA-GPV-VP3 DNA vaccine immunized geese respectively by the different routes of gene gun bombardment and intramuscular injection, which all could be detected in all tissues, and expressed VP3 in liver, ileum, cecum, heart, jejunum, duodenum, rectum, position of injected spot; pcDNA-GPV-VP3 DNA immunized by gene gun bombard- ment demonstrated higher copy numbers in all tissues and earlier expressed in liver, cecum than intramuscular injection at nonage; but at 217d post administration were no different.
5. Different dynamic distribution and expression of GPV-VP3 gene vaccine in gosling with the different doses: The quantity of pcDNA-GPV-VP3 and expression detected in the experimental groups showed the relations with the quantity of the inoculation doses, but not proportion (P≥0.05).
6. Infection and excretion of attenuated vaccine of GPV in gosling: Geese were immunized with live attenuated vaccineof GPV, the copy number decreased at the beginning then increased in 1h-3d, the maximum amount at 7d post administration, after 35d the copy number decreased gradually; At the position of injected spot, heart, liver and intestinal, viral antigens were detected in 1d-3d, the maximum amount at 7d; compared with the DNA vaccine, attenuated GPV vaccine was detected more copy number and expression in tissues, the difference was significant (P≤0.01≤0.05); but the number dropped quickly, all tissues failed to be detected antigen at 63d.
引文
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