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

发布时间：2018-06-26 05:15

本文选题：FQ-PCR + pcDNA-DEV-gC　；参考：《四川农业大学》2010年硕士论文

【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(P0.05)。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(P0.05),3d-18wk差异不显著(P0.05)。 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。
[Abstract]:In this paper, the following real-time fluorescent quantitative PCR (FQ-PCR) method was developed to detect the DNA of duck viral enteritis virus (DEV) by real-time fluorescence quantitative PCR (FQ-PCR), focusing on the dynamic distribution of the DNA of duck enteritis virus (DEV) in mice immunized with DEV GC gene vaccine. 2 DEV GC gene vaccine (pcDNA-DEV-gC) was bombarded with gene gun (6 渭 g / 3 渭 g / min), intramuscular injection (200 渭 g / 100 渭 g / g), intramuscular injection positive liposome / pcDNA-DEV-gC nanoparticles and chitosan / pcDNA-DEV-gC nanoparticles. The method of oral positive liposome / pcDNA-DEV-gC nanoparticles and chitosan / pcDNA-DEV-gC nanoparticles was used to immunize 4 week-old BALB / c mice. The immune sites (skin or muscle), heart, liver, spleen, lung, kidney, brain, thymus were taken at different time after immunization. The dynamic distribution of pcDNA-DEV-gC in BALB / C mice was detected by FQ-PCR method in duodenum, rectum and cecum. The results are as follows: 1. The FQ-PCR method has the advantages of high sensitivity, high specificity and good repeatability. The efficiency of standard curve amplification is 100. The correlation coefficient between the number of nucleic acid templates and the Ct value determined by FQ-PCR is 1.000, which has a good linear relationship. Using the equation YP3.321X 45.822, the unknown sample can be accurately quantified (Y = the logarithmic value of sample Ct value X = sample copy number), which can be used not only to study the dynamic distribution of pcDNA-DEV-gC in mice, but also to study the dynamic distribution of pcDNA-DEV-gC in mice. 2. The distribution of pcDNA-DEV-gC in mice: pcDNA-DEV-gC was detected in tissues and organs of mice immunized for 1 hour, and pcDNA-DEV-gC was detected in all tissues and organs of mice immunized with pcDNA-DEV-gC, gene gun bombardment group and intramuscular injection group. The highest level of immune sites was detected, The content of liver and spleen was the highest in intramuscular injection of liposome and oral liposome, and the highest in duodenum and rectum after intramuscular injection of chitosan and oral chitosan. By the time of 18wk, pcDNA-DEV-gC could still be detected in the tissues and organs of all immune groups, but the content of pcDNA-DEV-gC in most tissues and organs was about 102-104.3. The relationship between the different doses and the distribution: the general rule of the contents in the tissues of mice immunized with different doses of pcDNA-DEV-gC was 6 渭 g, 3 渭 g, 1 渭 g, 100 渭 g, 50 渭 g. There was a weak positive correlation between the dose of gene vaccine and the content of gene vaccine in mice tissues in the three immune dose groups bombarded by gene gun, and there was no significant difference between the different doses (P0.05). There was a positive correlation between the dose of gene vaccine and the content of gene vaccine in mice tissues after intramuscular injection of three doses (P0.05), and there was no significant difference (P0.05) between 3d-18wk (P0.05). The dose of gene vaccine bombarded by gene gun is very small, but the content detected in mouse tissue is similar to that detected by intramuscular injection. Liposomes and chitosan had their respective advantages. Liver and spleen were the largest tissues in liposome group and duodenum and rectum were the largest in chitosan group.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1

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