棉酚对新疆多浪羊IFN-γ基因表达影响及pcDNA3.1-IFN-γ真核表达载体的构建和淋巴细胞转染

发布时间：2018-03-07 12:05

本文选题：棉酚　切入点：IFN-γ　出处：《塔里木大学》2015年硕士论文　论文类型：学位论文

【摘要】：棉酚，又名棉毒素或者棉籽饼，是棉属植物内形成的一种多酚类化合物存在于植株各部分的褐色点状色素腺体中，而棉籽饼粕确是一种产量大，价格低廉，富含蛋白质的动物饲料，可就是因为棉酚的毒性限制了这种优良饲料的大规模的应用。无论是对反刍动物还是非反刍动物都有毒性，且呈剂量依赖性，，会对动物的大脑、心、肝、血管等器官造成损害，致使家畜流产。IFN-γ是由免疫细胞分泌的一种细胞因子，是一种重要的免疫调节因子，能调节和激活T淋巴细胞的活性，以应对外界抗原的侵入，维持机体的平衡。为了探讨棉酚对免疫系统的影响以及IFN-γ在棉酚作用于免疫系统中的作用，本实验以新疆多浪羊为实验对象，分离多浪羊的外周血淋巴细胞，通过不同浓度的棉酚与淋巴细胞体外共培养，利用荧光定量PCR的方法，检测IFN-γ的表达量的变化，结果，随着棉酚浓度的不断递增，IFN-γ的表达量也呈逐渐增长的趋势。利用PCR的方法扩增IFN-γ的CDS区，通过HindIII和BamHI对pMD18T-IFN-γ和pcDNA3.1进行双酶切，连接，转化，测序和序列分析，结果成功克隆出IFN-γ的CDS区，序列长度455bp，比对结果表明其与羊的IFN-γ序列的一致性达到99%以上。进一步分离外周血淋巴细胞，利用RPMI1640完全培养基体外培养淋巴细胞，待细胞进入对数期，利用电转染系统，将pcDNA3.1-IFN-γ真核表达载体转入淋巴细胞中，利用半定量的方法检测转染组与对照组的IFN-γ的表达量。结果表明，通过比较转染组和对照组IFN-γ的表达量，转染组明显高于对照组。
[Abstract]:Gossypol, also known as cotton toxin or cottonseed cake, is a polyphenolic compound formed in cotton plants. Protein-rich animal feed, however, is limited by the toxicity of gossypol to the large-scale use of this fine feed. Both ruminant and non-ruminant animals are toxic, and in a dose-dependent manner, it affects the brains and hearts of animals. The liver, blood vessels and other organs are damaged, causing livestock abortion. IFN- 纬 is a cytokine secreted by immune cells and is an important immunomodulating factor, which can regulate and activate the activity of T lymphocytes in response to the invasion of external antigens. In order to study the effect of gossypol on immune system and the effect of IFN- 纬 on immune system, the peripheral blood lymphocytes of Doulang sheep in Xinjiang were isolated. By co-culture of gossypol and lymphocytes in vitro, the expression of IFN- 纬 was detected by fluorescence quantitative PCR. The results showed that the expression of IFN- 纬 increased with the increasing of gossypol concentration. The CDS region of IFN- 纬 was amplified by PCR. PMD18T-IFN- 纬 and pcDNA3.1 were digested, ligated, transformed, sequenced and sequenced by HindIII and BamHI. The CDS region of IFN- 纬 was cloned successfully. The sequence length was 455bp. the result of alignment showed that the consistency of IFN- 纬 sequence with sheep was more than 99%. The peripheral blood lymphocytes were further isolated and cultured in vitro on RPMI1640 complete medium, the cells were in logarithmic phase, and the electrotransfection system was used. The eukaryotic expression vector pcDNA3.1-IFN- 纬 was transferred into lymphocytes, and the expression of IFN- 纬 in transfection group and control group was detected by semi-quantitative method. The results showed that the expression of IFN- 纬 in transfection group was significantly higher than that in control group by comparing the expression of IFN- 纬 between transfection group and control group.
【学位授予单位】：塔里木大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826.5

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