应用siRNAs抑制乙型脑炎病毒复制及乙型脑炎“自杀性”DNA疫苗研究

发布时间：2019-03-14 09:20

【摘要】：乙型脑炎(Japanese encephalitis，JE)(简称乙脑)，是由乙型脑炎病毒(Japanese encephalitis virus JEV)引起的一种严重危害人畜健康的虫媒病毒性疾病。该病对人类危害巨大，约三分之一的患者会死亡，存活的患者中有40％-70％会留下神经后遗症。乙脑也是危害养猪业的重大疫病之一，该病感染猪造成的主要经济损失是导致严重的繁殖障碍性疾病。目前对于该病还没有有效的治疗方法。RNAi是机体本身存在的天然抗病毒机制，为人们预防和治疗病毒感染开启了希望之门。本实验利用RNAi技术，从抑制病毒复制的角度，为乙脑的免疫预防开辟新途径。同时，本研究还开展了乙脑病毒主要免疫原性基因E和NS1的“自杀性”DNA疫苗研究，并在此基础上探讨了牛疱疹病毒Ⅰ型(BHV-1)的VP22蛋白对乙脑病毒“自杀性”DNA疫苗免疫效果的影响。主要研究内容如下： 1．siRNAs对乙脑病毒NS1基因表达的抑制以乙脑病毒的NS1基因为靶基因，设计合成了4个siRNA序列，并构建了各自的表达质粒(pS-NS1A，pS-NS1B，pS-NS1C，pS-NS1D)；构建了NS1基因与EGFP融合的真核表达质粒(pCDNS1-EGFP)，并实现了二者在Vero细胞和BHK-21细胞中的融合表达；将siRNA表达质粒与pCDNS1-EGFP共转染细胞后，经荧光观察、流式细胞仪检测、Western blot、RT-PCR等方法证实：在4个siRNAs序列中，pS-NS1A的抑制作用不明显，而pS-NS1B、pS-NS1C、pS-NS1D对NS1基因表达有显著的特异性抑制作用，其中pS-NS1C和pS-NS1D抑制效果达到80％—90％。 2．siRNAs对乙脑病毒复制的抑制将上述构建的4个siRNAs表达质粒分别转染BHK-21细胞，24小时后感染130PFU乙脑病毒，经间接免疫荧光观察、Western blot、空斑形成实验、RT-PCR等方法证实：在4个siRNAs序列中，pS-NS1A的抑制作用不明显，pS-NS1B，，pS-NS1C，pS-NS1D对乙脑病毒复制有显著的特异性抑制作用，其中pS-NS1C，pS-NS1D抑制效果达到79％—86％。 3．乙脑病毒E、NS1基因“自杀性”DNA疫苗与常规DNA疫苗的免疫效果比较将乙脑病毒E和NS1基因分别插入到“自杀性”DNA疫苗载体—PSCAI(pS)，分别构建了pSNS1、pSE两种“自杀性”DNA疫苗。在此基础上，将BHV-1的VP22蛋白基因分别插入到NS1、E基因的上游，使VP22与NS1、E基因融合表达，从而构建了另两种“自杀性”DNA疫苗：pSBNS1、pSBE。间接免疫荧光实验证实所构建的质粒均在BHK-21细胞中获得表达。小鼠的动物试验表明：NS1基因的“自杀性”DNA疫苗可以诱导显著高于常规DNA疫苗的ELISA抗体和细胞免疫水平；E基因的“自杀性”DNA疫苗与常规DNA疫苗相比，在诱导体液免疫方面没有优势，
[Abstract]:Encephalitis B (Japanese encephalitis,JE) is an insect-borne disease caused by Japanese encephalitis virus (Japanese encephalitis virus JEV), which is harmful to the health of human and animal. The disease does great harm to humans, with about 1/3 of patients dying, and 40% and 70% of surviving patients with neurological sequelae. Japanese encephalitis (je) is one of the most important infectious diseases in pig industry. The main economic loss caused by the infection is serious reproductive disorders. RNAi is a natural antiviral mechanism which opens the door of hope for the prevention and treatment of viral infection. In this study, the RNAi technique was used to inhibit the replication of the virus and to open up a new way for the immunoprevention of Japanese encephalitis. At the same time, we also carried out the "suicide" DNA vaccine study of the major immunogenic genes E and NS1 of Japanese encephalitis virus (JEV). The effect of VP22 protein of bovine herpesvirus type 1 (BHV-1) on the immune response of Japanese encephalitis virus (JEV) suicide DNA vaccine was also studied. The main contents are as follows: four siRNA sequences were designed and synthesized by using the NS1 gene of Japanese encephalitis virus (JEV) as the target gene to inhibit the expression of NS1 gene in Japanese encephalitis virus (JEV) induced by 1.siRNAs. The expression plasmids (pS-NS1A,pS-NS1B,pS-NS1C,pS-NS1D) were constructed. The eukaryotic expression plasmid (pCDNS1-EGFP) of the fusion of NS1 gene and EGFP was constructed, and the fusion expression of the two genes in Vero cells and BHK-21 cells was realized. After co-transfection of siRNA expression plasmid with pCDNS1-EGFP, the results of fluorescence observation, flow cytometry and Western blot,RT-PCR showed that the inhibitory effect of pS-NS1A was not obvious in the four siRNAs sequences, while pS-NS1B,pS-NS1C, was not obvious. PS-NS1D significantly inhibited the expression of NS1 gene, and the inhibitory effect of pS-NS1C and pS-NS1D reached 80% and 90% respectively. 2.siRNAs inhibited the replication of Japanese encephalitis virus (JEV). The above four siRNAs expression plasmids were transfected into BHK-21 cells and infected with 130PFU je virus 24 hours later., Western blot, was observed by indirect immunofluorescence. Plaque formation test, RT-PCR and other methods confirmed that pS-NS1A had no obvious inhibitory effect on the replication of Japanese encephalitis virus (JEV) in the four siRNAs sequences, and pS-NS1B,pS-NS1C,pS-NS1D had a specific inhibitory effect on the replication of Japanese encephalitis virus (JEV). Among them, pS-NS1C, had a specific inhibitory effect. The inhibitory effect of pS-NS1D was 79% and 86% respectively. 3. Comparison of immune efficacy of Japanese encephalitis virus E, NS1 gene "suicide" DNA vaccine and conventional DNA vaccine to insert Japanese encephalitis virus E and NS1 genes into "suicide" DNA vaccine vector-PSCAI (pS), respectively. Two "suicide" DNA vaccines of pSNS1,pSE were constructed respectively. On this basis, the VP22 protein gene of BHV-1 was inserted into the upstream of NS1,E gene, and the fusion expression of VP22 and NS1,E gene was made. Two other "suicide" DNA vaccines, pSBNS1,pSBE., were constructed. Indirect immunofluorescence assay confirmed that the constructed plasmids were expressed in BHK-21 cells. The results of animal experiments in mice showed that the suicidal DNA vaccine of NS1 gene could induce significantly higher ELISA antibody and cellular immune level than that of conventional DNA vaccine. The "suicide" DNA vaccine of the E gene has no advantage in inducing humoral immunity compared with the conventional DNA vaccine.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392;S852.5

【相似文献】