本文關(guān)鍵詞：雞黑素瘤差異化相關(guān)基因5啟動(dòng)子的克隆及功能驗(yàn)證　出處：《廣西大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 雞 天然免疫 RLRs MDA5 啟動(dòng)子 RIG-I 干擾素 病毒

【摘要】：天然免疫是機(jī)體抵抗病原體入侵的第一道屏障,能夠利用模式識(shí)別受體(Pattern recognition receptors,PRRs)識(shí)別病原體相關(guān)分子模式(Pathogen associated molecular pattern,PAMP)。模式識(shí)別受體可分為toll樣受體(Toll-like receptors,TLRs)、核苷酸結(jié)合寡聚化結(jié)構(gòu)域蛋白樣受體(Nucleotide-binding oligomerization domain protein-like receptors,NLRs)和視黃酸誘導(dǎo)基因 Ⅰ 樣受體(Retinoicacid-inducible gene Ⅰ(RIG-I)-like receptors,RLRs)等受體家族。RLR受體家族包括視黃酸誘導(dǎo)基因Ⅰ(RIG-I)、黑素瘤差異化相關(guān)基因 5(Melanoma differentiation-associated gene 5,MDA5)和實(shí)驗(yàn)室遺傳與生理學(xué)蛋白2(Laboratory of genetics and physiology 2,LGP2)。這些家族成員在對(duì)胞內(nèi)的RNA病毒識(shí)別過程中起著重要作用,并能夠觸發(fā)一系列抗病毒天然免疫反應(yīng)。在哺乳動(dòng)物中,RIG-I和MDA5能夠識(shí)別不同但又有重疊的RNA病毒種類。RIG-I主要識(shí)別不同長度的、具有5'端三磷酸基團(tuán)或者二磷酸基團(tuán)的雙鏈RNA病毒,或長度小于1 kb的短鏈人工合成病毒RNA類似物聚肌苷酸-聚胞苷酸(Polyinosinic-polycytidylic acid,poly(I:C))雙鏈 RNA 病毒;MDA5則主要識(shí)別大于1 kb的長的、雙鏈小核糖核酸病毒和長鏈poly(I:C)。然而,MDA5的配體尚未完全明確。RIG-I在雞的基因組中是缺失的,這可能是相對(duì)于表達(dá)RIG-I基因的鴨和其他哺乳動(dòng)物,雞對(duì)某些特定的病原體更易感的原因。因此,在雞上,MDA5對(duì)細(xì)胞質(zhì)中的RNA病毒的識(shí)別及抗病毒天然免疫反應(yīng)的觸發(fā)顯得尤為重要。但是,目前雞MDA5基因的相關(guān)研究還較少,其表達(dá)的分子調(diào)控機(jī)制尚未明確。為此,在本研究中,我們克隆并分析MDA5全長啟動(dòng)子序列,研究其在病毒和非病毒物質(zhì)刺激時(shí)的活性狀態(tài),進(jìn)而為利用此啟動(dòng)子制備轉(zhuǎn)基因抗病雞種奠定基礎(chǔ)。具體研究結(jié)果如下:1.本研究以白羽雞基因組DNA為模板進(jìn)行PCR擴(kuò)增,成功克隆出長度約為2.5 kb的雞MDA5全長啟動(dòng)子序列。經(jīng)預(yù)測(cè),這段啟動(dòng)子區(qū)域中含有多個(gè)轉(zhuǎn)錄因子結(jié)合位點(diǎn),包括Sp1、GATA-1、TGGCA-結(jié)合蛋白、AP-1、AP-3、ER-alpha、T3R-alpha、C/EBP alpha、NF-1(-樣蛋白)和NF-E4的結(jié)合位點(diǎn);為了探討雞與其他物種在進(jìn)化上的關(guān)系,本研究將此啟動(dòng)子和其他鳥類、魚和哺乳動(dòng)物的啟動(dòng)子作了進(jìn)化樹分析。結(jié)果顯示,就MDA5啟動(dòng)子而言,雞與其他鳥類的進(jìn)化關(guān)系較近,與哺乳動(dòng)物的進(jìn)化關(guān)系較遠(yuǎn)。2.本研究構(gòu)建了此啟動(dòng)子的報(bào)告載體質(zhì)粒piggybac-MDA5-DsRed,并用此質(zhì)粒對(duì)雞DF-1細(xì)胞進(jìn)行轉(zhuǎn)染。經(jīng)過藥物篩選,本研究得到了穩(wěn)定轉(zhuǎn)染質(zhì)粒 piggybac-MDA5-DsRed 的細(xì)胞系,命名為 Piggybac-MDA5-DsRed 細(xì)胞系。在此細(xì)胞系中,DsRed的表達(dá)情況代表著MDA5啟動(dòng)子的活性。通過熒光顯微鏡觀察和流式細(xì)胞儀分析,本研究發(fā)現(xiàn):在正常生理?xiàng)l件下,表達(dá)DsRed的細(xì)胞非常少,幾乎為零,表明MDA5啟動(dòng)子的活性極低;然而,當(dāng)細(xì)胞受到長鏈poly(I:C)(1 kb)和短鏈poly(I:C)(1 kb)、干擾素-β(Interferon β,IFN-β)和雞傳染性法氏囊病毒的刺激(Infectious bursal disease virus,IBDV)時(shí),表達(dá)DsRed的細(xì)胞數(shù)量顯著上升,分別達(dá)到了 18%和18.0%、9.86%、2.01%和1.01%,說明MDA5啟動(dòng)子的活性被極大地激活了。實(shí)時(shí)定量PCR結(jié)果顯示,細(xì)胞在受到poly(I:C)、IFN-β和IBDV的刺激時(shí),DsRed的mRNA水平均得到顯著的提高,與內(nèi)源性MDA5和IFN-β的mRNA水平變化趨勢(shì)是一致的。由于DsRed的mRNA水平代表MDA5啟動(dòng)子的活性,故本研究中的啟動(dòng)子活性與內(nèi)源性MDA5的表達(dá)水平是一致的,啟動(dòng)子活性可以反應(yīng)內(nèi)源性MDA5的表達(dá)情況。此外,本研究的結(jié)果顯示,相對(duì)于短鏈poly(I:C),長鏈poly(I:C)刺激能誘導(dǎo)更多細(xì)胞表達(dá)DsRed,引起更大的mRNA水平變化,說明相對(duì)于短鏈poly(I:C),雞MDA5能夠更好地識(shí)別長鏈poly(I:C)。同時(shí),長鏈poly(I:C)的刺激不僅能夠促進(jìn)細(xì)胞內(nèi)IFN-β表達(dá),還能夠促進(jìn)細(xì)胞內(nèi)IFN-α表達(dá),而短鏈poly(I:C)刺激未能引起IFN-α表達(dá)量的變化,故推測(cè)長鏈poly(I:C)和短鏈poly(I:C)引導(dǎo)的下游信號(hào)通路略有差異。3.為了驗(yàn)證雞MDA5啟動(dòng)子在雞轉(zhuǎn)基因抗病育種中的應(yīng)用價(jià)值,本研究以PB轉(zhuǎn)座子為背景,構(gòu)建了由雞MDA5啟動(dòng)子啟動(dòng)表達(dá)鴨RIG-I基因的重組表達(dá)載體,命名為PB-chMDA5-duRIG-I-mkate。試驗(yàn)證明,在正常情況下,檢測(cè)不到鴨RIG-I的表達(dá);而在病毒模擬物poly(I:C)刺激后,以此質(zhì)粒為依托的鴨RIG-I能夠在雞DF-1細(xì)胞中表達(dá)。本研究首次成功克隆出了雞MDA5啟動(dòng)子并對(duì)其功能進(jìn)行了初步研究。本研究的結(jié)果表明,此啟動(dòng)子與得到的Piggybac-MDA5-DsRed細(xì)胞系可以用來檢測(cè)雞MDA5的配體,驗(yàn)證其能否調(diào)節(jié)內(nèi)源性MDA5基因的表達(dá)。此外,由于此啟動(dòng)子的活性在正常生理?xiàng)l件下極低,但又在外源刺激物的作用下極大地提高,因此可以被用來啟動(dòng)表達(dá)其他抗病毒蛋白等目的蛋白,具有較好的應(yīng)用價(jià)值。本研究對(duì)雞天然免疫系統(tǒng)中的重要模式識(shí)別受體MDA5的啟動(dòng)子進(jìn)行初步探索,將為針對(duì)雞MDA5基因表達(dá)調(diào)控機(jī)制的研究提供理論依據(jù),有利于完善人們對(duì)雞天然免疫系統(tǒng)的認(rèn)知。同時(shí),本研究構(gòu)建的鴨RIG-I表達(dá)載體質(zhì)粒PB-chMDA5-duRIG-I-mkate,為表達(dá)RIG-I的轉(zhuǎn)基因雞的生產(chǎn)奠定了基礎(chǔ),進(jìn)而為生產(chǎn)抗病毒轉(zhuǎn)基因雞開辟新了的道路。
[Abstract]:Innate immunity is the first barrier against pathogen invasion in the organism, can use pattern recognition receptors (Pattern, recognition receptors, PRRs) recognition of pathogen associated molecular patterns (Pathogen associated molecular pattern, PAMP). Pattern recognition receptors can be divided into toll like receptors (Toll-like, receptors, TLRs) nucleotide binding oligomerization domain protein receptor (Nucleotide-binding oligomerization domain protein-like receptors, NLRs) and retinoic acid inducible gene 1 receptor (Retinoicacid-inducible gene 1 (RIG-I) -like receptors, RLRs) and.RLR receptor family receptors including retinoic acid inducible gene 1 (RIG-I), melanoma differentiation associated gene 5 (Melanoma differentiation-associated 5 gene, MDA5) and genetic laboratory with the physiology of protein 2 (Laboratory of genetics and physiology 2, LGP2). The family members in the RNA plays an important role in the process of intracellular virus identification, and can trigger a series of antiviral innate immune responses. In mammals, RIG-I and MDA5 can identify different but have different length of RNA virus type.RIG-I mainly recognize overlapping, double stranded RNA virus with 5'terminal three phosphate groups or two phosphate groups short chain, synthetic analogues of RNA virus or less than 1 kb in length polyinosinic polycytidylic acid (Polyinosinic-polycytidylic, acid, poly (I:C)) double stranded RNA virus; MDA5 is mainly identified more than 1 KB long, small double stranded RNA viruses and long chain poly (I:C). However, MDA5 the ligand.RIG-I is not completely clear in the chicken genome is missing, which may be relative to the expression of RIG-I gene of duck and other mammals, chicken to certain pathogens are more susceptible. Therefore, in the chicken, MDA5 of RN in the cytoplasm Identification and antiviral innate immune response to A virus trigger is particularly important. However, the related research of chicken MDA5 gene is still less, the expression of the molecular regulation mechanism is not clear. Therefore, in this study, we cloned and analyzed the full-length MDA5 promoter sequence, to study the viral and non viral substances when stimulated the active state, and the promoter of preparation of disease resistant transgenic chicken to lay the foundation. The results are as follows: 1. in this study, white chicken genome DNA as template for PCR amplification, clone a length of about 2.5 KB of the chicken MDA5 full-length promoter sequence. After the forecast, contains a number of transcription factor binding sites this section of the promoter region including Sp1, GATA-1, TGGCA- binding protein, AP-1, AP-3, ER-alpha, T3R-alpha, C/EBP, alpha, NF-1 (- like protein) and NF-E4 binding sites; in order to investigate the chicken and other species in evolution. In this study the relationship between promoter and other birds, fish and mammalian promoter as phylogenetic analysis. The results showed that the MDA5 promoter, close evolutionary relationship of chicken and other birds and mammals, the evolutionary relationship is far.2. the establishment of this promoter reporter plasmid piggybac-MDA5-DsRed. And the chicken DF-1 cells were transfected with this plasmid. After drug screening, this study obtained the stable transfection of plasmid piggybac-MDA5-DsRed cell line, named Piggybac-MDA5-DsRed cells. This cell line, the expression of DsRed represents the situation of MDA5 promoter activity. By fluorescence microscopy and flow cytometric analysis, this study found: under normal physiological conditions, the expression of DsRed is very small, almost zero, showed that MDA5 promoter activity is very low; however, when cells were long chain poly (I:C) (1 KB) and short Chain poly (I:C) (1 KB), interferon beta (Interferon beta, beta IFN-) and infectious bursal disease virus (Infectious bursal disease virus, stimulation, IBDV) expression significantly increased the number of DsRed cells, respectively 18% and 18%, 9.86%, 2.01% and 1.01%, said that MDA5 start the activity was greatly activated. Real time quantitative PCR results showed that cells in response to poly (I:C), IFN- beta and IBDV stimulation, DsRed mRNA levels were significantly improved, is consistent with the change of mRNA level of endogenous MDA5 and IFN- beta trend. Due to the DsRed level of mRNA on behalf of MDA5 promoter the expression level of activity, so the study of promoter activity and endogenous MDA5 is consistent with the expression of promoter activity can react with endogenous MDA5. In addition, the results of this study show that, compared with short chain poly (I:C), long chain poly (I:C) stimulation can induce more cells expressing Ds Red, mRNA level changes caused by the larger, relative to that of short chain poly (I:C), chicken MDA5 better able to identify long chain poly (I:C). At the same time, the long chain poly (I:C) stimulation can promote the expression of IFN- beta cells, but also can promote the expression of IFN- in cells, while the short chain poly (I:C) stimulation failed to induce changes in the expression of IFN- alpha, it is speculated that the long chain and short chain poly (I:C) poly (I:C) signaling pathways downstream of slight differences in the.3. guide in order to verify the application value of chicken MDA5 promoter in chicken transgenic breeding. In this study, PB transposon background, construct promoter expression vector expression of duck RIG-I gene by recombinant chicken MDA5 promoter, named as PB-chMDA5-duRIG-I-mkate. proof test, under normal circumstances, the expression of duck RIG-I was not detected; and the simulation of poly in virus (I:C) after stimulation with this plasmid based on duck RIG-I can be expressed in DF-1 cells in the chicken. The first study successfully cloned the chicken MDA5 promoter and its function were studied. The results of this study suggest that this ligand promoter and Piggybac-MDA5-DsRed cell lines can be used to detect the expression of chicken MDA5, verify whether the regulation of endogenous MDA5 gene. In addition, the promoter activity in normal physiological conditions under very low, but the exogenous stimulus greatly improved, therefore can be used to initiate the expression of other antiviral protein to protein, and has good application value. This study explores the promoter of important pattern recognition receptors of the innate immune system of chicken MDA5, provide a theoretical basis for the research the chicken MDA5 gene expression regulation mechanism, is conducive to improve the cognition of the chicken innate immune system. At the same time, this study constructed RIG-I expression vector plasmid PB-chMDA5-duRI duck G-I-mkate, which lays the foundation for the production of transgenic chicken with RIG-I, opens a new way for the production of antiviral transgenic chicken.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S858.31

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