本文選題：FQ-PCR + pcDNA-DEV-gC��； 參考：《四川農(nóng)業(yè)大學(xué)》2010年碩士論文

【摘要】： 本論文圍繞鴨病毒性腸炎病毒(DEV) gC基因檢測方法、DEV gC基因疫苗免疫小鼠后在機體的動態(tài)分布開展了下列研究：①檢測DEV gC基因的實時熒光定量PCR (FQ-PCR)方法的建立。②將DEV gC基因疫苗(pcDNA-DEV-gC)分別以基因槍轟擊法(6μg／只、3μg／只、1μg／只),肌肉注射法(200μg／只、100μg／只、50gg／只),肌肉注射陽性脂質(zhì)體/pcDNA-DEV-gC納米粒和殼聚糖/pcDNA-DEV-gC納米粒,口服陽性脂質(zhì)體/pcDNA-DEV-gC納米粒和殼聚糖/pcDNA-DEV-gC納米粒的方法免疫4周齡BALB/c小鼠,免疫后不同時間分別取各組小鼠的免疫部位(皮膚或肌肉)、心臟、肝臟、脾臟、肺臟、腎臟、腦、胸腺、十二指腸、直腸和盲腸等組織器官,用建立的FQ-PCR的方法檢測pcDNA-DEV-gC在BALB/C小鼠體內(nèi)的動態(tài)分布。獲得如下結(jié)果： 1.建立的FQ-PCR檢測方法靈敏度高、特異性強、重復(fù)性好,標準曲線擴增效率為100%,核酸模板數(shù)與FQ-PCR測定的Ct值相關(guān)系數(shù)達到1.000,具有很好的線性關(guān)系,使用方程Y=-3.321X+45.822能夠?qū)ξ粗獦悠愤M行精確定量(Y=樣品Ct值,X=樣品拷貝數(shù)的對數(shù)值),不僅能夠用于研究pcDNA-DEV-gC在小鼠體內(nèi)的動態(tài)分布,而且可以用于DEV的臨床快速檢測。 2. pcDNA-DEV-gC在小鼠體內(nèi)的分布規(guī)律：各免疫組免疫小鼠1h即可在各組織器官中檢測到pcDNA-DEV-gC,基因槍轟擊組和肌肉注射組檢測到免疫部位含量最高,其次是肝臟、脾臟和胸腺,肌肉注射脂質(zhì)體和口服脂質(zhì)體檢測到肝臟和脾臟含量最高,肌肉注射殼聚糖和口服殼聚糖檢測到十二指腸和直腸含量最高。到18wk時,各免疫組各個組織器官內(nèi)仍然能夠檢測到pcDNA-DEV-gC的存在,但多數(shù)組織器官中的含量比免疫1h時降低了約102-104。 3.不同免疫劑量與分布的關(guān)系：不同劑量pcDNA-DEV-gC免疫小鼠各組織中的含量呈現(xiàn)的總體規(guī)律為6μg組3μg組1μg組,200μg組100μg組50μg組。在基因槍轟擊三個免疫劑量組中,基因疫苗的劑量與基因疫苗在小鼠組織中的含量呈現(xiàn)較弱的正相關(guān)性,各劑量之間差異不顯著(P0.05)。在肌肉注射三個免疫劑量組中,基因疫苗的劑量與基因疫苗在小鼠組織中的含量呈現(xiàn)正相關(guān)性,1h-3d差異顯著(P0.05),3d-18wk差異不顯著(P0.05)。 4.基因槍轟擊基因疫苗劑量很小,但在小鼠組織中檢測出的含量與肌肉注射相似。脂質(zhì)體和殼聚糖各有優(yōu)勢,脂質(zhì)體組檢測含量最大的組織是肝臟和脾臟,殼聚糖組檢測含量最大的組織是十二指腸和直腸。
[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.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.1

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