本文選題：雞　切入點(diǎn)：內(nèi)源性反轉(zhuǎn)錄病毒　出處：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：內(nèi)源性反轉(zhuǎn)錄病毒(Endogenous retrovirus.ERV)作為基因組成分,其生物學(xué)功能正在被逐漸認(rèn)識(shí)。近年來(lái)的研究表明在哺乳動(dòng)物中內(nèi)源性反轉(zhuǎn)錄病毒的表達(dá)受營(yíng)養(yǎng)因素和表遺傳的調(diào)控,而內(nèi)源性反轉(zhuǎn)錄病毒可通過(guò)調(diào)控先天免疫相關(guān)的轉(zhuǎn)錄因子來(lái)參與先天免疫反應(yīng)。然而,有關(guān)家禽的ERV研究還相當(dāng)有限。比如,家禽營(yíng)養(yǎng)因子是否調(diào)節(jié)ERV的表達(dá),ERV是否受表遺傳調(diào)控,營(yíng)養(yǎng)因子是否通過(guò)調(diào)控ERV來(lái)影響動(dòng)物的先天免疫反應(yīng)等。本研究將圍繞這些問(wèn)題進(jìn)行研究,試圖闡明營(yíng)養(yǎng)因子對(duì)雞ERV表達(dá)的影響及相關(guān)機(jī)制。主要研究結(jié)果如下:1、雞內(nèi)源性反轉(zhuǎn)錄病毒ALVE1基因序列及其表達(dá)分析:利用鏈特異性引物,以雞巨噬細(xì)胞HD11基因組為模板進(jìn)行ALVE1全長(zhǎng)序列PCR分段擴(kuò)增,測(cè)序結(jié)果表明克隆測(cè)序的ALVE1基因序列與NCBI公布的ALVE1序列同源性達(dá)到99%,但多個(gè)位點(diǎn)存在核苷酸多態(tài)性。利用熒光定量PCR檢測(cè)ALVE1在CEF細(xì)胞和HD11細(xì)胞的轉(zhuǎn)錄表達(dá)以及ALVE1 env基因在2日齡和35日齡SPF雞不同組織(腦、法氏囊、胸腺、脾臟、心臟、肝臟、腎臟、肺臟和皮膚)中的表達(dá)量,結(jié)果顯示:env基因在免疫細(xì)胞HD11中的表達(dá)量顯著高于非免疫細(xì)胞CEF(P0.05);在2日齡雞免疫器官脾臟中的表達(dá)量也高于其他組織(P0.05),而且2日齡SPF雞免疫器官(脾臟和法氏囊)中env的表達(dá)量顯著高于35日齡的SPF雞(P0.05)。以上結(jié)果提示ALVE1可能參與雞早期免疫器官的形成及早期先天免疫反應(yīng)。不過(guò),該推測(cè)還有待進(jìn)一步研究加以驗(yàn)證。2、營(yíng)養(yǎng)因子對(duì)ALVE1表達(dá)的影響在培養(yǎng)基中分別用蛋氨酸和維生素C處理雞細(xì)胞,然后用qPCR檢測(cè)ALVE1和先天免疫基因的表達(dá)量。結(jié)果表明,蛋氨酸激活了雞內(nèi)源性反轉(zhuǎn)錄病毒LTR序列的轉(zhuǎn)錄,并伴有先天免疫相關(guān)基因表達(dá)的顯著上長(zhǎng)升(P0.05)。然而,維生素C處理對(duì)ALVE1的轉(zhuǎn)錄表達(dá)沒(méi)有顯著影響(P0.05)。3、ALVE1區(qū)CpG到的甲基化分析生物信息學(xué)預(yù)測(cè)發(fā)現(xiàn)ALVE1基因存在兩個(gè)CpG甲基化島,且ALVE1 env轉(zhuǎn)錄物存在miR-155作用位點(diǎn)。DNA甲基化酶抑制劑AZA處理HD11細(xì)胞后,焦磷酸測(cè)序的結(jié)果表明:ALVE1 CpG島的DNA甲基化水平降低,且伴有ALVE1基因和先天免疫相關(guān)基因的表達(dá)激活。再者,利用Overlap PCR以及熒光素酶報(bào)告系統(tǒng),研究證實(shí)miR-155可靶向env的轉(zhuǎn)錄物。以上結(jié)果表明:ALVE1的表達(dá)受表觀遺傳學(xué)機(jī)制的調(diào)控,且ALVE1的表達(dá)激活可能參與先天免疫反應(yīng)。另外,通過(guò)脂質(zhì)體轉(zhuǎn)染ALVE1 env基因,使其過(guò)表達(dá)近40倍,但并未對(duì)所測(cè)先天免疫基因的表達(dá)產(chǎn)生顯著影響,這可能是反義RNA表達(dá)激活的缺失所致。綜上所述,雞內(nèi)源性反轉(zhuǎn)錄病毒ALVE1 env基因在雞免疫細(xì)胞以及免疫器官中高表達(dá),特別是雞的早期生長(zhǎng)階段。ALVE1的轉(zhuǎn)錄表達(dá)受到蛋氨酸、miR-155以及DNA甲基化的調(diào)控。營(yíng)養(yǎng)因子-蛋氨酸可能通過(guò)誘導(dǎo)ALVE1的表達(dá)來(lái)激活先天免疫基因的表達(dá),影響動(dòng)物的免疫反應(yīng)。這些研究結(jié)果可能對(duì)推動(dòng)內(nèi)源性反轉(zhuǎn)錄病毒的表觀遺傳學(xué)研究具有重要意義。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65

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