Development Of TaqMan FQ-PCR Assay Based On DEV GC Gene And Studies On Dynamic Distribution Of GC Gene Vaccine In Mice

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.