本文選題：RNAi + Mx��； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：豬流感(Swine influenza,SI)是由豬流感病毒(Swine influenza virus,SIV)引起的一種急性、高度接觸性的群發(fā)性呼吸道傳染病,能夠造成豬群免疫抑制,給養(yǎng)豬業(yè)帶來(lái)重大危害,同時(shí)會(huì)給人類健康構(gòu)成威脅,具有重要的公共衛(wèi)生意義。RNA干擾(RNA inierference,RNAi)是在進(jìn)化過(guò)程中高度保守、由雙鏈RNA(double-stranded RNA,dsRNA)誘發(fā)的高效特異性地降解同源mRNA的現(xiàn)象,能特異抑制病毒基因的表達(dá);Mx蛋白是I型(α/β)和III型(λ)干擾素(IFN)誘導(dǎo)產(chǎn)生的細(xì)胞自身天然抗病毒活性的關(guān)鍵效應(yīng)分子,是一種廣譜的抗病毒蛋白�，F(xiàn)代養(yǎng)豬生產(chǎn),生產(chǎn)性狀與抗性性狀之間存在一定程度的遺傳拮抗,單純追求高產(chǎn)目標(biāo)通常導(dǎo)致豬抵抗力降低;通過(guò)轉(zhuǎn)基因技術(shù)改造豬的遺傳性狀,提高機(jī)體免疫力,可能是控制傳染性疾病的有效手段。本實(shí)驗(yàn)室前期篩選出RNAi、Mx和4shRNA三個(gè)基因,并在細(xì)胞水平或小鼠體內(nèi)或者靜脈豬體內(nèi)證實(shí)證明三個(gè)基因具有抗病價(jià)值。在此基礎(chǔ)上本研究探索將這三個(gè)基因聯(lián)合構(gòu)建到慢病毒載體中,包裝慢病毒后感染豬胎兒成纖維細(xì)胞制備核供體細(xì)胞,通過(guò)體細(xì)胞核移植技術(shù)成功獲得F0代克隆豬。運(yùn)用分子生物學(xué)方法PCR、Southern blot、RT-PCR、Western blot分別在DNA、RNA、蛋白質(zhì)水平對(duì)F0代豬進(jìn)行鑒定分析,結(jié)果表明,3頭F0代克隆豬均為陽(yáng)性轉(zhuǎn)基因豬;選取其中一頭與同品種野生型豬(WT)進(jìn)行雜交,繁育F1代轉(zhuǎn)基因豬。利用上述分子生物學(xué)方法對(duì)F1代轉(zhuǎn)基因豬進(jìn)行分析,結(jié)果表明4頭F1代轉(zhuǎn)基因豬中1頭有外源基因的整合和表達(dá),為陽(yáng)性轉(zhuǎn)基因豬。證明表達(dá)4shRNA-Mx-RNAi多基因轉(zhuǎn)基因豬轉(zhuǎn)入的外源基因能夠穩(wěn)定遺傳至F1代。為進(jìn)一步探究F1代轉(zhuǎn)基因豬成纖維細(xì)胞的抑制流感病毒復(fù)制效果,本研究分離培養(yǎng)1日齡F1代轉(zhuǎn)基因豬尾組織成纖維細(xì)胞,100TCID50 A/Swine/Guangdong/2004(H1N1)毒株感染后,通過(guò)間接免疫熒光(IFA)、實(shí)時(shí)熒光定量PCR(Real-time PCR)、病毒滴度測(cè)定分析了F1代轉(zhuǎn)基因豬在細(xì)胞水平上抑制流感病毒復(fù)制的活性。結(jié)果表明,在攻毒后不同時(shí)間點(diǎn),與非轉(zhuǎn)基因豬和WT豬相比,轉(zhuǎn)基因豬成纖維細(xì)胞能夠抑制流感病毒增殖;感染流感病毒后各時(shí)間點(diǎn),外源基因都有一定的抑制流感病毒的活性,而在16h時(shí)抑制效率達(dá)到最高,IFA結(jié)果顯示轉(zhuǎn)基因豬成纖維細(xì)胞對(duì)流感病毒的抑制效率高于非轉(zhuǎn)基因豬和野生型豬,細(xì)胞中H1N1 PB2基因復(fù)制效率比WT降低2倍,細(xì)胞上清中病毒TCID50達(dá)1×10-2.8。表明表達(dá)多基因轉(zhuǎn)基因豬在細(xì)胞水平上具有抑制流感病毒復(fù)制的活性。本研究成功構(gòu)建了表達(dá)4shRNA-Mx-RNAi重組慢病毒載體,利用體細(xì)胞核移植技術(shù)制備了能夠表達(dá)多基因抗流感轉(zhuǎn)基因豬,并證實(shí)其體內(nèi)有外源基因的整合與表達(dá),能夠穩(wěn)定遺傳至F1代,其成纖維細(xì)胞具有抑制流感病毒復(fù)制活性,為多基因協(xié)同抑制流感病毒復(fù)制研究提供了轉(zhuǎn)基因豬模型,為探索新的抑制流感病毒復(fù)制策略和轉(zhuǎn)基因動(dòng)物抗病研究奠定基礎(chǔ)。
[Abstract]:Swine influenza influenzae sil (SIV) is an acute, highly contagious mass respiratory infectious disease caused by swine flu virus Swine influenza virus. It can cause immunosuppression in pigs, cause serious harm to pig industry and pose a threat to human health. RNAi is a highly conserved, highly efficient and specific degradation of homologous mRNA induced by double-stranded RNA(double-stranded RNAs. Mx protein, which can specifically inhibit the expression of virus gene, is a key effector molecule of natural antiviral activity of cells induced by type I (偽 / 尾) and III type (位) interferon. It is a broad-spectrum antiviral protein. In modern pig production, there is a certain degree of genetic antagonism between production traits and resistance traits. May be an effective means of controlling infectious diseases. Three 4shRNA genes were screened in our laboratory and proved to be resistant to the disease at the cell level, in mice or in intravenous pigs. On this basis, this study explored the construction of these three genes into lentivirus vector, packaging lentivirus infected porcine fetal fibroblasts to prepare nuclear donor cells, and successfully obtained F0 generation cloned pig by somatic cell nuclear transfer technology. Molecular biology method was used to identify and analyze F0 generation pigs by Western blot. The results showed that all of the three cloned pigs were positive transgenic pigs, and one of them was crossbred with wild type pigs of the same breed. Breeding F 1 transgenic pigs. The results showed that one of the four F1 transgenic pigs had the integration and expression of exogenous genes and was positive for transgenic pigs. The results showed that the foreign genes expressed in 4shRNA-Mx-RNAi polygene transgenic pigs could be stably inherited to F1 generation. In order to further investigate the inhibitory effect of F1 generation transgenic porcine fibroblasts on influenza virus replication, we isolated and cultured 100 TCID50 A / Swine- / Guangdong/ 2004 H1 / N1 virus strain after 1 day old F1 generation transgenic porcine tail tissue fibroblasts were isolated and cultured. Indirect immunofluorescence assay (IFA) and real-time fluorescence quantitative PCR(Real-time PCR assay were used to determine the viral titer of F1 transgenic pigs in inhibiting influenza virus replication at cell level. The results showed that compared with non-transgenic pigs and WT pigs, the fibroblasts of transgenic pigs could inhibit the proliferation of influenza virus at different time points after infection. The results of IFA showed that the inhibitory efficiency of transgenic pig fibroblasts against influenza virus was higher than that of non-transgenic pigs and wild type pigs. The replication efficiency of H1N1 PB2 gene in cells was 2 times lower than that of WT, and the virus TCID50 in the supernatant was 1 脳 10 ~ (- 2) -2.8%. The results showed that transgenic pigs expressing polygene had the ability to inhibit influenza virus replication at cell level. In this study, the recombinant lentivirus vector expressing 4shRNA-Mx-RNAi was successfully constructed, and the multigene anti-influenza transgenic pigs were prepared by using somatic cell nuclear transfer technique, and the integration and expression of exogenous genes were confirmed, which could be inherited to F1 generation stably. The fibroblasts have the ability to inhibit the replication of influenza virus, which provides a transgenic pig model for the study of co-inhibition of influenza virus replication by polygenes, and lays a foundation for the exploration of new strategies for inhibiting influenza virus replication and the study of disease resistance of transgenic animals.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

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