本文關鍵詞： 熒光定量PCR IL-4 IL-10 IL-13 IFN-α 免疫評價 犬三聯(lián)活疫苗　出處：《東北農業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：實時熒光定量PCR技術是在PCR反應體系中加入熒光探針或者熒光染料,利用熒光信號的積累,實時監(jiān)測整個PCR的反映進程,通過制作出的標準曲線,對未知模版進行定量分析。因其具有操作簡便、快速高效、高敏感性等特點,被廣泛應用于分子診斷、分子生物研究、動物及食品安全檢測等方面。本實驗分離了犬全血中的外周血淋巴細胞,通過PCR成功擴增出IL-4、IL-10、IL-13、IFN-α等幾種細胞免疫因子的基因片段,連接pMD-18T載體,轉化到DH5α感受態(tài)細胞中,在Amp抗性培養(yǎng)基上進行篩選、PCR和測序等驗證,構建了標準品質粒。最后成功建立了檢測犬全血中IL-4、IL-10、IL-13、IFN-α等細胞因子的實時熒光定量PCR的方法,該方法的模版在102~108拷貝/μL范圍內呈現(xiàn)良好的線性關系,相關系數(shù)R2均達到0.992以上。利用該方法對犬瘟熱病毒(CDV)、犬細小病毒(CPV)、犬腺病毒Ⅰ型(CAV-1)三聯(lián)活疫苗免疫比格犬后的細胞免疫水平進行了評價。實驗結果顯示:犬三聯(lián)活疫苗免疫犬后,免疫犬外周血淋巴細胞與空白對照外周血淋巴細胞相比,IL-4、IL-10、IL-13、IFN-α等幾種細胞免疫因子mRNA水平在3~14天之間均顯著升高(p0.05);空白對照組的4種細胞免疫因子mRNA水平?jīng)]有明顯差異(p0.05)。以上實驗結果表明,本研究建立的實時熒光定量PCR方法確實可行,IL-4、IL-10、IL-13、IFN-α等細胞免疫因子可以作為細胞免疫評價指標之一。
[Abstract]:Real-time fluorescence quantitative PCR technology is to add fluorescent probes or fluorescent dyes into the PCR reaction system, using the accumulation of fluorescence signals, real-time monitoring the whole PCR reaction process, through the preparation of the standard curve. Quantitative analysis of unknown templates has been widely used in molecular diagnosis and molecular biology research because of its advantages of simple operation, high efficiency, high sensitivity and so on. The peripheral blood lymphocytes were isolated from the whole blood of dogs, and IL-4 IL-10 IL-13 was amplified successfully by PCR. The gene fragments of IFN- 偽 and other cytokines were ligated into pMD-18T vector and transformed into DH5 偽 competent cells, and screened on Amp resistant medium. The standard plasmid was constructed by PCR and sequencing. Finally, the detection of IL-4, IL-10 and IL-13 in the whole blood of dogs was successfully established. IFN- 偽 and other cytokines of real-time fluorescence quantitative PCR method, the template of this method in 102108copies / 渭 L showed a good linear relationship. The correlation coefficient R2 was above 0.992. The method was used to treat canine distemper virus (CDV) and canine parvovirus (CPV). The cellular immunity level of Beagle dogs immunized with CAV-1 triple live vaccine was evaluated. The results showed that the immunized dogs were immunized with three live vaccines. The peripheral blood lymphocytes of immunized dogs were compared with the peripheral blood lymphocytes of blank control. The level of IFN- 偽 and other cytokines mRNA increased significantly between 3d and 14d. There was no significant difference in the levels of mRNA among the four cytokines in the blank control group. The above results showed that the real-time fluorescent quantitative PCR method established in this study is feasible for IL-4. Cytokines such as IL-10, IL-13, IFN- 偽 can be used as one of the indexes for evaluating cellular immunity.
【學位授予單位】：東北農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.4

【共引文獻】

相關期刊論文 前10條

1 翁善鋼;陸明輝;王晏罡;;犬傳染性氣管支氣管炎相關的呼吸道病毒研究概況[J];廣東畜牧獸醫(yī)科技;2012年06期

2 王玉龍,華育平;狐腦炎病毒研究進展[J];經(jīng)濟動物學報;2004年02期

3 張洋;袁子國;姜秋杰;劉曄;崔艷;張守峰;扈榮良;;犬2型腺病毒的分離鑒定及初步特性分析[J];軍事醫(yī)學科學院院刊;2008年06期

4 孔令雯;董競成;;氧化應激致組蛋白修飾在慢性阻塞性肺病發(fā)生發(fā)展中的作用[J];醫(yī)學研究生學報;2014年09期

5 劉曄;房麗君;趙敬慧;張守峰;張菲;扈榮良;;鼬獾狂犬病病毒分離株的培養(yǎng)及其免疫原性[J];中國生物制品學雜志;2011年02期

6 葛艷華;姜騫;劉家森;司昌德;郭東春;林歡;曲連東;;犬腺病毒2型抗原的制備及間接ELISA的建立[J];實驗動物科學;2010年05期

7 劉錦宙;楊燕;謝而付;;STAT6-ORF表達載體的構建與鑒定[J];中國生化藥物雜志;2014年08期

8 王建科;程世鵬;易立;楊莘;羅彬;許紅麗;閆喜軍;武華;;水貂腸炎病毒雙抗體夾心ELISA檢測方法的建立[J];中國獸醫(yī)科學;2011年02期

9 劉大飛;姜一f，

本文編號：1479108