基于DEV gC基因FQ-PCR方法建立及gC基因疫苗在免疫小鼠体内分布规律的研究
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摘要
本论文围绕鸭病毒性肠炎病毒(DEV) gC基因检测方法、DEV gC基因疫苗免疫小鼠后在机体的动态分布开展了下列研究：①检测DEV gC基因的实时荧光定量PCR (FQ-PCR)方法的建立。②将DEV gC基因疫苗(pcDNA-DEV-gC)分别以基因枪轰击法(6μg／只、3μg／只、1μg／只),肌肉注射法(200μg／只、100μg／只、50gg／只),肌肉注射阳性脂质体/pcDNA-DEV-gC纳米粒和壳聚糖/pcDNA-DEV-gC纳米粒,口服阳性脂质体/pcDNA-DEV-gC纳米粒和壳聚糖/pcDNA-DEV-gC纳米粒的方法免疫4周龄BALB/c小鼠,免疫后不同时间分别取各组小鼠的免疫部位(皮肤或肌肉)、心脏、肝脏、脾脏、肺脏、肾脏、脑、胸腺、十二指肠、直肠和盲肠等组织器官,用建立的FQ-PCR的方法检测pcDNA-DEV-gC在BALB/C小鼠体内的动态分布。获得如下结果：
1.建立的FQ-PCR检测方法灵敏度高、特异性强、重复性好,标准曲线扩增效率为100%,核酸模板数与FQ-PCR测定的Ct值相关系数达到1.000,具有很好的线性关系,使用方程Y=-3.321X+45.822能够对未知样品进行精确定量(Y=样品Ct值,X=样品拷贝数的对数值),不仅能够用于研究pcDNA-DEV-gC在小鼠体内的动态分布,而且可以用于DEV的临床快速检测。
2. pcDNA-DEV-gC在小鼠体内的分布规律：各免疫组免疫小鼠1h即可在各组织器官中检测到pcDNA-DEV-gC,基因枪轰击组和肌肉注射组检测到免疫部位含量最高,其次是肝脏、脾脏和胸腺,肌肉注射脂质体和口服脂质体检测到肝脏和脾脏含量最高,肌肉注射壳聚糖和口服壳聚糖检测到十二指肠和直肠含量最高。到18wk时,各免疫组各个组织器官内仍然能够检测到pcDNA-DEV-gC的存在,但多数组织器官中的含量比免疫1h时降低了约102-104。
3.不同免疫剂量与分布的关系：不同剂量pcDNA-DEV-gC免疫小鼠各组织中的含量呈现的总体规律为6μg组>3μg组>1μg组,200μg组>100μg组>50μg组。在基因枪轰击三个免疫剂量组中,基因疫苗的剂量与基因疫苗在小鼠组织中的含量呈现较弱的正相关性,各剂量之间差异不显著(P>0.05)。在肌肉注射三个免疫剂量组中,基因疫苗的剂量与基因疫苗在小鼠组织中的含量呈现正相关性,1h-3d差异显著(P<0.05),3d-18wk差异不显著(P>0.05)。
4.基因枪轰击基因疫苗剂量很小,但在小鼠组织中检测出的含量与肌肉注射相似。脂质体和壳聚糖各有优势,脂质体组检测含量最大的组织是肝脏和脾脏,壳聚糖组检测含量最大的组织是十二指肠和直肠。
In this thesis, research about the method of detecting gC gene of duck enteritis virus (DEV), the dynamic distribution of DEV gC gene vaccine in mice were as follows:①The TaqManTM fluorescent quantitative real-time PCR (FQ-PCR) method for detecting DEV gC gene was developed.②The pcDNA-DEV-gC vaccine was vaccinated into ten groups of BALB/c mice (4 weeks old) by gene gun bombing with three doses (6μg per mouse,3μg per mouse and 1μg per mouse), intramuscularly injection with three doses (200μg per mouse,100μg per mouse and 50μg per mouse), intramuscularly injection with liposomes/chitosan microparticles and oral with liposomes/chitosan microparticles, then injected spot (skin or muscle), hreat, liver, spleen, lung, kidney, brain, thymus, duodemun, rectum and cecum of mice were collected at sampling times and the established FQ-PCR method was used to detect the dynamic distribution of pcDNA-DEV-gC vaccine in tissues of mice. Below the results were show:
1. the developed FQ-PCR method was a highly specific, sensitive, repeatable and reproducible assay, it displayed a clear linear relationship with a correlation coefficient of 1.000 and high amplification efficiency (100%).By using the following formula, we were able to quantify the amount of unknown samples:Y=-3.321X+ 45.822 (Y=threshold cycle, X=log starting quantity).
2. The dynamic distribution of DEV gC gene vaccine in mice:DEV gC can be detected in all analyzed tissues at 1 hour postinoculation. the copies was most at the position of injected spot, then liver spleen and thymus in groups of gene gun bombing and intramuscularly injection, the copies was most at the position of liver and spleen in groups of intramuscularly injection with liposomes/chitosan microparticles, the copies was most at the position of duodemun and rectum in groups of oral with liposomes/chitosan microparticles. pcDNA-DEV-gC could still be detected in tissues of different doses groups in 18 weeks postinoculation. But the copies decreased about 102-104copies in the most tissues than it at 1 hour postinoculation.
3. Different dynamic distribution of DEV gC gene vaccine in mice with the different doses:in the three dose groups of gene gun bombing, there was a weak positive correiation between the immune doses and the copies of the tissues, the difference among the three groups was notsignificant (P>0.05). In the three dose groups of intramuscularly injection, there was a positive correiation between the immune doses and the copies of the tissues, the difference among the three groups was significant from 1 hour to 3 days postinoculation (P<0.05) and notsignificant from 5 days to 18 weeks postinoculation (P>0.05).
4. The dose of gene gun bombing was very small, but the copies in it were similar with the copies in groups of intramuscularly injection. The liposomes and chitosan microparticles have some advantages, the copies was most at the position of liver and spleen in groups of liposomes, then the copies was most at the position of duodemun and rectum in groups of chitosan microparticles.
1.建立的FQ-PCR检测方法灵敏度高、特异性强、重复性好,标准曲线扩增效率为100%,核酸模板数与FQ-PCR测定的Ct值相关系数达到1.000,具有很好的线性关系,使用方程Y=-3.321X+45.822能够对未知样品进行精确定量(Y=样品Ct值,X=样品拷贝数的对数值),不仅能够用于研究pcDNA-DEV-gC在小鼠体内的动态分布,而且可以用于DEV的临床快速检测。
2. pcDNA-DEV-gC在小鼠体内的分布规律：各免疫组免疫小鼠1h即可在各组织器官中检测到pcDNA-DEV-gC,基因枪轰击组和肌肉注射组检测到免疫部位含量最高,其次是肝脏、脾脏和胸腺,肌肉注射脂质体和口服脂质体检测到肝脏和脾脏含量最高,肌肉注射壳聚糖和口服壳聚糖检测到十二指肠和直肠含量最高。到18wk时,各免疫组各个组织器官内仍然能够检测到pcDNA-DEV-gC的存在,但多数组织器官中的含量比免疫1h时降低了约102-104。
3.不同免疫剂量与分布的关系：不同剂量pcDNA-DEV-gC免疫小鼠各组织中的含量呈现的总体规律为6μg组>3μg组>1μg组,200μg组>100μg组>50μg组。在基因枪轰击三个免疫剂量组中,基因疫苗的剂量与基因疫苗在小鼠组织中的含量呈现较弱的正相关性,各剂量之间差异不显著(P>0.05)。在肌肉注射三个免疫剂量组中,基因疫苗的剂量与基因疫苗在小鼠组织中的含量呈现正相关性,1h-3d差异显著(P<0.05),3d-18wk差异不显著(P>0.05)。
4.基因枪轰击基因疫苗剂量很小,但在小鼠组织中检测出的含量与肌肉注射相似。脂质体和壳聚糖各有优势,脂质体组检测含量最大的组织是肝脏和脾脏,壳聚糖组检测含量最大的组织是十二指肠和直肠。
In this thesis, research about the method of detecting gC gene of duck enteritis virus (DEV), the dynamic distribution of DEV gC gene vaccine in mice were as follows:①The TaqManTM fluorescent quantitative real-time PCR (FQ-PCR) method for detecting DEV gC gene was developed.②The pcDNA-DEV-gC vaccine was vaccinated into ten groups of BALB/c mice (4 weeks old) by gene gun bombing with three doses (6μg per mouse,3μg per mouse and 1μg per mouse), intramuscularly injection with three doses (200μg per mouse,100μg per mouse and 50μg per mouse), intramuscularly injection with liposomes/chitosan microparticles and oral with liposomes/chitosan microparticles, then injected spot (skin or muscle), hreat, liver, spleen, lung, kidney, brain, thymus, duodemun, rectum and cecum of mice were collected at sampling times and the established FQ-PCR method was used to detect the dynamic distribution of pcDNA-DEV-gC vaccine in tissues of mice. Below the results were show:
1. the developed FQ-PCR method was a highly specific, sensitive, repeatable and reproducible assay, it displayed a clear linear relationship with a correlation coefficient of 1.000 and high amplification efficiency (100%).By using the following formula, we were able to quantify the amount of unknown samples:Y=-3.321X+ 45.822 (Y=threshold cycle, X=log starting quantity).
2. The dynamic distribution of DEV gC gene vaccine in mice:DEV gC can be detected in all analyzed tissues at 1 hour postinoculation. the copies was most at the position of injected spot, then liver spleen and thymus in groups of gene gun bombing and intramuscularly injection, the copies was most at the position of liver and spleen in groups of intramuscularly injection with liposomes/chitosan microparticles, the copies was most at the position of duodemun and rectum in groups of oral with liposomes/chitosan microparticles. pcDNA-DEV-gC could still be detected in tissues of different doses groups in 18 weeks postinoculation. But the copies decreased about 102-104copies in the most tissues than it at 1 hour postinoculation.
3. Different dynamic distribution of DEV gC gene vaccine in mice with the different doses:in the three dose groups of gene gun bombing, there was a weak positive correiation between the immune doses and the copies of the tissues, the difference among the three groups was notsignificant (P>0.05). In the three dose groups of intramuscularly injection, there was a positive correiation between the immune doses and the copies of the tissues, the difference among the three groups was significant from 1 hour to 3 days postinoculation (P<0.05) and notsignificant from 5 days to 18 weeks postinoculation (P>0.05).
4. The dose of gene gun bombing was very small, but the copies in it were similar with the copies in groups of intramuscularly injection. The liposomes and chitosan microparticles have some advantages, the copies was most at the position of liver and spleen in groups of liposomes, then the copies was most at the position of duodemun and rectum in groups of chitosan microparticles.
引文
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