蛋氨酸对鸡内源性反转录病毒ALVE1表达的影响及其相关调控机制研究

发布时间：2018-03-26 09:56

本文选题：鸡　切入点：内源性反转录病毒　出处：《扬州大学》2017年硕士论文

【摘要】：内源性反转录病毒(Endogenous retrovirus.ERV)作为基因组成分,其生物学功能正在被逐渐认识。近年来的研究表明在哺乳动物中内源性反转录病毒的表达受营养因素和表遗传的调控,而内源性反转录病毒可通过调控先天免疫相关的转录因子来参与先天免疫反应。然而,有关家禽的ERV研究还相当有限。比如,家禽营养因子是否调节ERV的表达,ERV是否受表遗传调控,营养因子是否通过调控ERV来影响动物的先天免疫反应等。本研究将围绕这些问题进行研究,试图阐明营养因子对鸡ERV表达的影响及相关机制。主要研究结果如下:1、鸡内源性反转录病毒ALVE1基因序列及其表达分析:利用链特异性引物,以鸡巨噬细胞HD11基因组为模板进行ALVE1全长序列PCR分段扩增,测序结果表明克隆测序的ALVE1基因序列与NCBI公布的ALVE1序列同源性达到99%,但多个位点存在核苷酸多态性。利用荧光定量PCR检测ALVE1在CEF细胞和HD11细胞的转录表达以及ALVE1 env基因在2日龄和35日龄SPF鸡不同组织(脑、法氏囊、胸腺、脾脏、心脏、肝脏、肾脏、肺脏和皮肤)中的表达量,结果显示:env基因在免疫细胞HD11中的表达量显著高于非免疫细胞CEF(P0.05);在2日龄鸡免疫器官脾脏中的表达量也高于其他组织(P0.05),而且2日龄SPF鸡免疫器官(脾脏和法氏囊)中env的表达量显著高于35日龄的SPF鸡(P0.05)。以上结果提示ALVE1可能参与鸡早期免疫器官的形成及早期先天免疫反应。不过,该推测还有待进一步研究加以验证。2、营养因子对ALVE1表达的影响在培养基中分别用蛋氨酸和维生素C处理鸡细胞,然后用qPCR检测ALVE1和先天免疫基因的表达量。结果表明,蛋氨酸激活了鸡内源性反转录病毒LTR序列的转录,并伴有先天免疫相关基因表达的显著上长升(P0.05)。然而,维生素C处理对ALVE1的转录表达没有显著影响(P0.05)。3、ALVE1区CpG到的甲基化分析生物信息学预测发现ALVE1基因存在两个CpG甲基化岛,且ALVE1 env转录物存在miR-155作用位点。DNA甲基化酶抑制剂AZA处理HD11细胞后,焦磷酸测序的结果表明:ALVE1 CpG岛的DNA甲基化水平降低,且伴有ALVE1基因和先天免疫相关基因的表达激活。再者,利用Overlap PCR以及荧光素酶报告系统,研究证实miR-155可靶向env的转录物。以上结果表明:ALVE1的表达受表观遗传学机制的调控,且ALVE1的表达激活可能参与先天免疫反应。另外,通过脂质体转染ALVE1 env基因,使其过表达近40倍,但并未对所测先天免疫基因的表达产生显著影响,这可能是反义RNA表达激活的缺失所致。综上所述,鸡内源性反转录病毒ALVE1 env基因在鸡免疫细胞以及免疫器官中高表达,特别是鸡的早期生长阶段。ALVE1的转录表达受到蛋氨酸、miR-155以及DNA甲基化的调控。营养因子-蛋氨酸可能通过诱导ALVE1的表达来激活先天免疫基因的表达,影响动物的免疫反应。这些研究结果可能对推动内源性反转录病毒的表观遗传学研究具有重要意义。
[Abstract]:As a genome component, the biological function of endogenous retrovirus (Endogenous retrovirus.ERV) is being gradually recognized. Recent studies have shown that the expression of endogenous retrovirus in mammals is regulated by nutritional and epigenetic factors. Endogenous retroviruses can participate in innate immune responses by regulating transcription factors associated with innate immunity. However, ERV studies on poultry are limited. For example, Whether the poultry nutrition factor regulates the expression of ERV and whether it is regulated by epigenetic regulation, whether the nutrition factor affects the innate immune response of animals by regulating ERV, etc. This paper attempts to elucidate the effect of nutritional factors on chicken ERV expression and its related mechanisms. The main results are as follows: 1. The sequence and expression of ALVE1 gene of chicken endogenous retrovirus were analyzed by using chain specific primers. Chicken macrophage HD11 genome was used as template to amplify the full-length ALVE1 sequence PCR. Sequencing results showed that the sequence of ALVE1 gene cloned and sequenced was 99% homology to the ALVE1 sequence published by NCBI, but nucleotide polymorphisms existed at several sites. The transcriptional expression of ALVE1 and ALVE1 in CEF and HD11 cells were detected by fluorescence quantitative PCR. Env gene was expressed in different tissues (brain) of SPF chickens at the age of 2 and 35 days. In the bursa of Fabricius, thymus, spleen, heart, liver, kidney, lung and skin. The results showed that the expression of the gene in HD11 of immune cells was significantly higher than that of non-immune cells, and the expression level in spleen of 2-day-old chicken was also higher than that of other tissues, and the immune organs (spleen and bursa of Fabricius) of 2-day-old SPF chickens were also higher than those of other tissues. The expression of env in SPF chickens was significantly higher than that in SPF chickens at 35 days of age. The above results suggested that ALVE1 might be involved in the formation of early immune organs and the early innate immune response. The effect of nutritional factors on the expression of ALVE1 in the culture medium was treated with methionine and vitamin C, and then the expression of ALVE1 and innate immune genes were detected by qPCR. Methionine activates the transcription of the LTR sequence of the chicken endogenous retrovirus and is accompanied by a significant increase in the expression of innate immune-associated genes (P0.05). There was no significant effect of vitamin C treatment on the transcriptional expression of ALVE1. The bioinformatics analysis of CpG from P0.05 to Ave 1 region predicted that there were two CpG methylation islands in the ALVE1 gene. Furthermore, ALVE1 env transcripts had miR-155 action sites. AZA, a depressant of miR-155 methylase, treated HD11 cells. The results of pyrosequencing showed that the DNA methylation level in HD11 cells was decreased and the expression of ALVE1 gene and innate immune-related genes were activated. Using Overlap PCR and luciferase reporting system, it has been proved that miR-155 can target env transcripts. These results indicate that the expression of miR-155 is regulated by epigenetic mechanism, and the activation of ALVE1 expression may be involved in innate immune response. Transfection of ALVE1 env gene by liposome makes it overexpression nearly 40 times, but it has no significant effect on the expression of innate immune gene, which may be due to the lack of activation of antisense RNA expression. Chicken endogenous retrovirus ALVE1 env gene is highly expressed in chicken immune cells and immune organs. In particular, the transcriptional expression of ALVE1 was regulated by methionine miR-155 and DNA methylation. The nutritional factor-methionine may activate the expression of innate immune genes by inducing the expression of ALVE1. These results may play an important role in the epigenetic study of endogenous retroviruses.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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