多基因遗传修饰猪体外抑制猪流感病毒增殖的初步分析

发布时间：2018-05-13 04:34

本文选题：RNAi + Mx　；参考：《中国农业科学院》2015年硕士论文

【摘要】：猪流感(Swine influenza,SI)是由猪流感病毒(Swine influenza virus,SIV)引起的一种急性、高度接触性的群发性呼吸道传染病,能够造成猪群免疫抑制,给养猪业带来重大危害,同时会给人类健康构成威胁,具有重要的公共卫生意义。RNA干扰(RNA inierference,RNAi)是在进化过程中高度保守、由双链RNA(double-stranded RNA,dsRNA)诱发的高效特异性地降解同源mRNA的现象,能特异抑制病毒基因的表达;Mx蛋白是I型(α/β)和III型(λ)干扰素(IFN)诱导产生的细胞自身天然抗病毒活性的关键效应分子,是一种广谱的抗病毒蛋白。现代养猪生产,生产性状与抗性性状之间存在一定程度的遗传拮抗,单纯追求高产目标通常导致猪抵抗力降低;通过转基因技术改造猪的遗传性状,提高机体免疫力,可能是控制传染性疾病的有效手段。本实验室前期筛选出RNAi、Mx和4shRNA三个基因,并在细胞水平或小鼠体内或者静脉猪体内证实证明三个基因具有抗病价值。在此基础上本研究探索将这三个基因联合构建到慢病毒载体中,包装慢病毒后感染猪胎儿成纤维细胞制备核供体细胞,通过体细胞核移植技术成功获得F0代克隆猪。运用分子生物学方法PCR、Southern blot、RT-PCR、Western blot分别在DNA、RNA、蛋白质水平对F0代猪进行鉴定分析,结果表明,3头F0代克隆猪均为阳性转基因猪;选取其中一头与同品种野生型猪(WT)进行杂交,繁育F1代转基因猪。利用上述分子生物学方法对F1代转基因猪进行分析,结果表明4头F1代转基因猪中1头有外源基因的整合和表达,为阳性转基因猪。证明表达4shRNA-Mx-RNAi多基因转基因猪转入的外源基因能够稳定遗传至F1代。为进一步探究F1代转基因猪成纤维细胞的抑制流感病毒复制效果,本研究分离培养1日龄F1代转基因猪尾组织成纤维细胞,100TCID50 A/Swine/Guangdong/2004(H1N1)毒株感染后,通过间接免疫荧光(IFA)、实时荧光定量PCR(Real-time PCR)、病毒滴度测定分析了F1代转基因猪在细胞水平上抑制流感病毒复制的活性。结果表明,在攻毒后不同时间点,与非转基因猪和WT猪相比,转基因猪成纤维细胞能够抑制流感病毒增殖;感染流感病毒后各时间点,外源基因都有一定的抑制流感病毒的活性,而在16h时抑制效率达到最高,IFA结果显示转基因猪成纤维细胞对流感病毒的抑制效率高于非转基因猪和野生型猪,细胞中H1N1 PB2基因复制效率比WT降低2倍,细胞上清中病毒TCID50达1×10-2.8。表明表达多基因转基因猪在细胞水平上具有抑制流感病毒复制的活性。本研究成功构建了表达4shRNA-Mx-RNAi重组慢病毒载体,利用体细胞核移植技术制备了能够表达多基因抗流感转基因猪,并证实其体内有外源基因的整合与表达,能够稳定遗传至F1代,其成纤维细胞具有抑制流感病毒复制活性,为多基因协同抑制流感病毒复制研究提供了转基因猪模型,为探索新的抑制流感病毒复制策略和转基因动物抗病研究奠定基础。
[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 (伪 / 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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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