本文選題：流感病毒 + RNA聚合酶陷阱系統(tǒng)��； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：流感病毒是引起動(dòng)物和人呼吸道疾病的重要病原體,對(duì)畜牧生產(chǎn)和公共衛(wèi)生安全構(gòu)成了巨大的威脅。控制流感病毒在畜禽中的感染和傳播,對(duì)畜牧生產(chǎn)和人類健康具有重要意義。隨著動(dòng)物轉(zhuǎn)基因技術(shù)的迅速發(fā)展,通過基因修飾的方法培育抗流感病毒的畜禽新品種,為在畜牧業(yè)中有效防治流感疫情提供了新的方法。流感病毒基因組末端具有保守的5'和3'非翻譯區(qū),特別是5'非翻譯區(qū)最末端的13個(gè)堿基在流感病毒基因組所有片段中全部相同,3'非翻譯區(qū)最末端的12個(gè)堿基只有一個(gè)位置的堿基會(huì)發(fā)生變化。保守的非翻譯區(qū)包含有流感病毒RNA復(fù)制和轉(zhuǎn)錄的順式作用元件,能夠被流感病毒聚合酶識(shí)別,在病毒的復(fù)制、轉(zhuǎn)錄和包裝中起到重要作用。本研究設(shè)計(jì)了一個(gè)流感病毒聚合酶陷阱系統(tǒng)(Influenza A Virus RNA Polymerase Decoy System, IVPDS),該系統(tǒng)載體能夠表達(dá)類似于流感病毒RNA (viral RNA like, vRNA-like)序列,該序列兩側(cè)帶有流感病毒基因組5'和3'非翻譯區(qū)。vRNA-like序列競(jìng)爭(zhēng)性結(jié)合流感病毒的聚合酶,一方面抑制聚合酶對(duì)流感病毒的復(fù)制,另一方面vRNA-like序列在聚合酶的作用下進(jìn)行復(fù)制和轉(zhuǎn)錄,表達(dá)外源蛋白,激活宿主的免疫反應(yīng),增加宿主對(duì)病毒的抗性。在本研究中,構(gòu)建了由RNA聚合酶Ⅰ啟動(dòng)子轉(zhuǎn)錄的表達(dá)GFP的IVPDS報(bào)告載體,pIVPDS-I-G。瞬時(shí)轉(zhuǎn)染pIVPDS-I-G的細(xì)胞或穩(wěn)定整合pIVPDS-I-G-Neo的細(xì)胞(簡(jiǎn)稱IVPDS-G細(xì)胞)都能夠轉(zhuǎn)錄出含有GFP的類似于流感病毒的序列vRNA-GFP(-)。在瞬轉(zhuǎn)pIVPDS-I-G或穩(wěn)定整合的IVPDS-G細(xì)胞中轉(zhuǎn)染病毒聚合酶和NP表達(dá)載體,檢測(cè)到了 mRNA-GFP(+)以及GFP蛋白的表達(dá)。流感病毒感染上述細(xì)胞后可以得到相似的結(jié)果,但與轉(zhuǎn)染病毒聚合酶和NP表達(dá)載體相比,細(xì)胞感染流感病毒后誘導(dǎo)表達(dá)的mRNA-GFP(+)以及GFP蛋白的表達(dá)量顯著降低。在轉(zhuǎn)染pIVPDS-I-G的DF-1細(xì)胞中,共轉(zhuǎn)流感病毒聚合酶和NP表達(dá)載體能提高I型干擾素以及I型干擾素下游基因mRNA水平,說明IVPDS載體被誘導(dǎo)后產(chǎn)生的雙鏈RNA能激活Ⅰ型干擾素通路。為了分析pIVPDS-I-G對(duì)流感病毒復(fù)制的影響,檢測(cè)了感染流感病毒的細(xì)胞內(nèi)病毒mRNA的表達(dá)量和細(xì)胞培養(yǎng)液中的病毒滴度。與空白對(duì)照組相比,瞬轉(zhuǎn)pIVPDS-I-G的細(xì)胞或IVPDS-G細(xì)胞內(nèi)流感病毒Martix基因mRNA水平均顯著降低,但是胞外上清中病毒滴度沒有顯著差異。此外,本研究中還構(gòu)建了表達(dá)人MxA和小鼠Mx (總稱Mx)的IVPDS載體,pIVPDS-I-Mx-Neo。在 293T 和 DF-1 細(xì)胞瞬時(shí)轉(zhuǎn)染 pIVPDS-I-Mx-Neo 能夠轉(zhuǎn)錄 vRNA-Mx(-),共轉(zhuǎn)染pIVPDS-I-Mx-Neo、流感病毒聚合酶和NP蛋白質(zhì)粒時(shí),檢測(cè)到細(xì)胞中mRNA-Mx(+)和Mx蛋白質(zhì)的表達(dá)。在瞬時(shí)轉(zhuǎn)染pIVPDS-I-Mx-Neo的DF-1細(xì)胞感染流感病毒后,檢測(cè)細(xì)胞上清中病毒滴度,結(jié)果顯示在24 hpi和36 hpi轉(zhuǎn)染pIVPDS-I-MxA-Neo的細(xì)胞上清中的病毒滴度顯著低于空載體對(duì)照組,轉(zhuǎn)染pIVPDS-I-Mx1-Neo的細(xì)胞上清中的病毒滴度與空載體對(duì)照組相比并沒有顯著的差異。綜上所述,本研究成功構(gòu)建了 IVPDS,該系統(tǒng)載體表達(dá)的vRNA-like序列能夠被流感病毒聚合酶識(shí)別進(jìn)行轉(zhuǎn)錄和翻譯,達(dá)到了誘導(dǎo)性表達(dá)外源基因的目的,轉(zhuǎn)染pIVPDS-I-MxA-Neo的細(xì)胞對(duì)流感病毒產(chǎn)生了一定抗性。本研究驗(yàn)證了流感病毒聚合酶陷阱系統(tǒng)抑制流感病毒復(fù)制策略的可行性,為下一步的研究和應(yīng)用該系統(tǒng)進(jìn)行抗流感病毒育種奠定基礎(chǔ)并提供了新的思路。
[Abstract]:Influenza virus is an important pathogen causing animal and human respiratory diseases, which poses a great threat to animal production and public health safety. Controlling infection and transmission of influenza virus in livestock and poultry is of great significance to animal production and human health. With the rapid development of transgenic techniques in animals, the gene modified Fang Fapei has been developed. A new breed of livestock and poultry resistant to influenza virus provides a new method for effective prevention and control of influenza in animal husbandry. The end of the genome of the influenza virus has a conservative 5'and 3' non translation area, especially the 13 bases at the most end of the 5'non translation region in all segments of the genome of the influenza virus genome, and the most terminal 12 base of the non translated region of the 3' The base of only one location changes. The conservative non translation area contains the cis acting element with the replication and transcription of influenza virus RNA, which can be identified by influenza virus polymerase and plays an important role in the replication, transcription and packaging of the virus. A influenza virus polymerase trap system (Influenza A Virus RNA P) is designed in this study. Olymerase Decoy System, IVPDS), the system vector can express the sequence of influenza virus RNA (viral RNA like, vRNA-like), which combines the polymerase of influenza virus genome 5'and 3' untranslated region.VRNA-like sequences, on the one hand inhibit the replication of the polymerase to influenza virus, on the other hand, -like sequences replicate and transcribe under the action of polymerase, express foreign proteins, activate the host immune response, and increase host resistance to the virus. In this study, a IVPDS reporter vector expressing GFP, a transcription of RNA polymerase I promoter, was constructed, pIVPDS-I-G. was transiently transfected to pIVPDS-I-G cells or stable integrated pIVPDS-I-G-Neo The cells (IVPDS-G cells) can transcribe the sequence vRNA-GFP (-) containing GFP, which are similar to influenza viruses. Virus polymerase and NP expression vectors are transfected into pIVPDS-I-G or stable IVPDS-G cells. The expression of mRNA-GFP (+) and GFP protein is detected. Influenza virus infection can be similar to those of the above cells. However, compared with the transfected virus polymerase and NP expression vector, the expression of mRNA-GFP (+) and GFP protein in the infected cells infected with influenza virus decreased significantly. In the DF-1 cells transfected with pIVPDS-I-G, the influenza virus polymerase and NP expression vector could improve the mRNA level of the I type interferon and the downstream gene of the type I interferon. In order to analyze the effect of pIVPDS-I-G on the replication of influenza virus, the expression of intracellular virus mRNA and the titer of virus in cell culture fluid were detected by the double stranded RNA induced by the induced IVPDS vector. In contrast to the blank control group, the transient pIVPDS-I-G cell or the intracellular flow of IVPDS-G cells was compared with that of the blank control group. The mRNA level of the Martix gene of the virus was significantly decreased, but there was no significant difference in the titer of the virus in the supernatant. In addition, the IVPDS vector expressing human MxA and mouse Mx (general Mx) was also constructed. The transient transfection of pIVPDS-I-Mx-Neo to pIVPDS-I-Mx-Neo. in 293T and DF-1 cells could transcribe vRNA-Mx (-) and co transfect pIVPDS-I-Mx-Neo, The expression of mRNA-Mx (+) and Mx protein in cells was detected when influenza virus polymerase and NP protein particles were detected. The virus titer in cell supernatant was detected after infected with influenza virus from DF-1 cells transfected instantaneously, and the results showed that the titer of the cell supernatant of 24 HPI and 36 HPI transfected pIVPDS-I-MxA-Neo was significantly lower than that of the empty body. In the control group, the virus titer in the cell supernatant transfected with pIVPDS-I-Mx1-Neo had no significant difference compared with the empty vector control group. In summary, the study successfully constructed the IVPDS. The vRNA-like sequence expressed by the system can be transcribed and translated by influenza virus polymerase, and the inducible expression of foreign genes is achieved. Objective: the transfected pIVPDS-I-MxA-Neo cells have a certain resistance to influenza virus. This study verified the feasibility of influenza virus polymerase trap system to inhibit the replication strategy of influenza virus, and provided a new idea for the next research and application of the system for the breeding of influenza virus.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 程從升;舒躍龍;張智清;;流感病毒的反向遺傳學(xué)研究進(jìn)展[J];病毒學(xué)報(bào);2007年01期

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本文編號(hào)：2052303