戊型肝炎病毒编码的microRNA-A6对病毒复制的影响
发布时间：2018-08-05 18:11
【摘要】：戊型肝炎病毒(Hepatitis E Virus, HEV)是一种经粪口途径传播,严重危害人类健康的病毒性肝炎的病原体。HEV属于肝炎病毒科,肝炎病毒属,无囊膜的单股正链RNA病毒,由三个开放阅读框(Open Reading Fragment, ORF)组成。HEV可以跨种间感染人和多种动物。在发展中国家,戊型肝炎发病率较高,对孕妇危害尤为严重,妊娠晚期孕妇感染HEV后死亡率高达25%。 目前,HEV复制机制尚不清楚,microRNA (miR)是一类进化上保守,在基因表达中起重要调控作用的小分子。病毒编码的miR是一类潜在的、小的、非抗原性的基因表达调节因子,可以通过调节宿主细胞免疫系统来创造适宜病毒复制的环境。]microRNA-A6(miR-A6)是HEV编码的其中一条miR,能够增强HEV抗原在动物体内的表达。本试验旨在细胞水平研究miR-A6对HEV复制的影响,为研究HEV复制机制提供基础。本研究工作包括3个部分： 1、miR-A6靶基因的筛选 利用TaregetScan5.1数据库搜索,结合"microRNA-靶基因”共调节统计学分析法筛选得到候选靶基因：SIRP-α(signal-regulatory protein alpha)、ING5(inhibitor of growth family, member5)和ZAP (zinc-finger antiviral protein)。将上述候选靶基因的3’UTR插入到含荧光素酶(Luciferase)报告基因的pMIR-REPORT载体中,构建候选靶基因报告载体。将三个候选靶基因报告载体分别和miR-A6类似物(miR-A6mimic)共转染A549细胞,转染24h后,荧光素酶实验证实miR-A6与SIRP-α、ING5和ZAP三个靶基因都具有相互作用。 2、miR-A6对候选靶基因的调控 构建靶基因与绿色荧光蛋白(EGFP)报告基因融合表达载体,分别命名为pcDNA3.1(+)/SIRP-a-EGFP、pcDNA3.1(+)/ING5-EGFP、pcDNA3.1(+)/ZAP-EGFP,分别转染HepG2细胞,通过观察荧光证实所构建的载体能在细胞中表达。miR-A6mimic分别与pcDNA3.1(+)/SIRP-α-EGFP、pcDNA3.1(+)/ING5-EGFP、pcDNA3.1(+)/ZAP-EGFP重组真核表达质粒共转染HEK293T细胞,利用Real-time qPCR技术检测miR-A6对靶基因在mRNA水平的调控作用,利用Western Blot方法检测miR-A6对靶基因在蛋白水平的调控作用。实验结果表明,miR-A6促进靶基因ZAP表达,抑制ING5和SIRP-α表达。 3、miR-A6对HEV复制的影响 MiR-A6mimic转染A549、HepG2细胞24h后接种HEV病毒样品,用含2%小牛血清的DMEM维持培养4天,通过免疫荧光方法检测HEV在细胞中的分布；PGC-A6和miR-A6抑制物(anti-A6)共转染A549、HepG2细胞24h后,接种HEV病毒样品,维持培养6天,收集细胞,运用Real-time qPCR方法检测niR-A6对HEV ORF1和ORF2mRNA转录的调控作用；使用Western Blot技术检测miR-A6对HEV ORF2蛋白表达的调控作用。研究表明,miR-A6对HEV无论在mRNA水平还是在蛋白水平都有增强作用。 本论文筛选得到3条候选靶基因,通过荧光素酶实验检测了候选靶基因与miR-A6有相互作用,Real-time qPCR和Western Blot技术验证了miR-A6可能通过对靶基因的调控作用影响HEV的复制。我们认为miR-A6可能通过调控靶基因,调节宿主天然免疫系统从而影响HEV的复制。本研究初步探索了HEV的复制机制,为HEV疫苗研究及抗病毒药物研发提供基础。
[Abstract]:Hepatitis E virus (Hepatitis E Virus, HEV) is a kind of viral hepatitis which is transmitted by the fecal path, which seriously endangers the human health of viral hepatitis,.HEV belongs to the hepatitis virus family, the hepatitis virus, the non cysts of single strand RNA virus, composed of three open reading frames (Open Reading Fragment, ORF), which can infect people and a variety of interspecies. Animals. In developing countries, the incidence of hepatitis E is high, especially for pregnant women. The mortality rate of pregnant women infected with HEV in the third trimester of pregnancy is as high as 25%.
At present, the mechanism of HEV replication is not yet clear. MicroRNA (miR) is a class of small molecules that have evolved conservatively and play an important role in gene expression. The miR encoded by the virus is a potential, small, non antigenic gene expression regulator, which can be used to create an environment suitable for the replication of the virus by regulating the host cell immune system. A6 (miR-A6) is one of the miR encoded by HEV, which can enhance the expression of HEV antigen in animals. This experiment aims to study the effect of miR-A6 on HEV replication and provide the basis for studying the mechanism of HEV replication. This study includes 3 parts:
Screening of target genes of 1, miR-A6
The candidate target genes were screened by TaregetScan5.1 database search and combined with the "microRNA- target gene" co regulated statistical analysis method: SIRP- alpha (signal-regulatory protein alpha), ING5 (inhibitor of growth family, member5) and signal-regulatory. In the pMIR-REPORT vector of the enzyme (Luciferase) reporter gene, the candidate target gene report carrier was constructed. The three candidate target gene reporter vectors were transfected to A549 cells with miR-A6 analogues (miR-A6mimic) respectively. After transfection of 24h, the luciferase test confirmed that both miR-A6 and SIRP- a, ING5 and ZAP three target genes had interaction.
Regulation of candidate target gene by 2, miR-A6
The fusion expression vector of target gene and green fluorescent protein (EGFP) reporter gene was constructed, named pcDNA3.1 (+) /SIRP-a-EGFP, pcDNA3.1 (+) /ING5-EGFP, pcDNA3.1 (+) /ZAP-EGFP, respectively, and HepG2 cells were transfected respectively. By observing the fluorescence, the vectors constructed could express.MiR-A6mimic respectively and pcDNA3.1 (+) /SIRP- alpha -EGFP, (+). /ING5-EGFP, pcDNA3.1 (+) /ZAP-EGFP recombinant eukaryotic expression plasmid co transfected HEK293T cells, and Real-time qPCR technique was used to detect the regulation effect of miR-A6 on the target gene at mRNA level, and Western Blot method was used to detect the regulatory role of miR-A6 on the protein level. SIRP- alpha expression.
3, the effect of miR-A6 on HEV replication
MiR-A6mimic transfected with A549, HepG2 cells were inoculated with HEV virus samples and cultured with DMEM containing 2% calf serum for 4 days. The distribution of HEV in the cells was detected by immunofluorescence. PGC-A6 and miR-A6 inhibitor (anti-A6) were co transfected to A549, HepG2 cells were inoculated for 6 days, and cells were collected and collected. The effects of niR-A6 on the transcription of HEV ORF1 and ORF2mRNA were detected, and Western Blot technique was used to detect the regulation of miR-A6 on the expression of ORF2 protein in HEV. The study showed that miR-A6 had an enhanced effect on HEV both in mRNA level and protein level.
3 candidate target genes were screened in this paper. The interaction between candidate target gene and miR-A6 was detected by luciferase test. Real-time qPCR and Western Blot showed that miR-A6 may affect the replication of HEV by regulating the target gene. We think miR-A6 may pass the regulation of target gene and regulate the host's natural immune system. This study explored the mechanism of HEV replication and provided the basis for HEV vaccine research and antiviral drug research.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R373.21
本文编号：2166584
[Abstract]:Hepatitis E virus (Hepatitis E Virus, HEV) is a kind of viral hepatitis which is transmitted by the fecal path, which seriously endangers the human health of viral hepatitis,.HEV belongs to the hepatitis virus family, the hepatitis virus, the non cysts of single strand RNA virus, composed of three open reading frames (Open Reading Fragment, ORF), which can infect people and a variety of interspecies. Animals. In developing countries, the incidence of hepatitis E is high, especially for pregnant women. The mortality rate of pregnant women infected with HEV in the third trimester of pregnancy is as high as 25%.
At present, the mechanism of HEV replication is not yet clear. MicroRNA (miR) is a class of small molecules that have evolved conservatively and play an important role in gene expression. The miR encoded by the virus is a potential, small, non antigenic gene expression regulator, which can be used to create an environment suitable for the replication of the virus by regulating the host cell immune system. A6 (miR-A6) is one of the miR encoded by HEV, which can enhance the expression of HEV antigen in animals. This experiment aims to study the effect of miR-A6 on HEV replication and provide the basis for studying the mechanism of HEV replication. This study includes 3 parts:
Screening of target genes of 1, miR-A6
The candidate target genes were screened by TaregetScan5.1 database search and combined with the "microRNA- target gene" co regulated statistical analysis method: SIRP- alpha (signal-regulatory protein alpha), ING5 (inhibitor of growth family, member5) and signal-regulatory. In the pMIR-REPORT vector of the enzyme (Luciferase) reporter gene, the candidate target gene report carrier was constructed. The three candidate target gene reporter vectors were transfected to A549 cells with miR-A6 analogues (miR-A6mimic) respectively. After transfection of 24h, the luciferase test confirmed that both miR-A6 and SIRP- a, ING5 and ZAP three target genes had interaction.
Regulation of candidate target gene by 2, miR-A6
The fusion expression vector of target gene and green fluorescent protein (EGFP) reporter gene was constructed, named pcDNA3.1 (+) /SIRP-a-EGFP, pcDNA3.1 (+) /ING5-EGFP, pcDNA3.1 (+) /ZAP-EGFP, respectively, and HepG2 cells were transfected respectively. By observing the fluorescence, the vectors constructed could express.MiR-A6mimic respectively and pcDNA3.1 (+) /SIRP- alpha -EGFP, (+). /ING5-EGFP, pcDNA3.1 (+) /ZAP-EGFP recombinant eukaryotic expression plasmid co transfected HEK293T cells, and Real-time qPCR technique was used to detect the regulation effect of miR-A6 on the target gene at mRNA level, and Western Blot method was used to detect the regulatory role of miR-A6 on the protein level. SIRP- alpha expression.
3, the effect of miR-A6 on HEV replication
MiR-A6mimic transfected with A549, HepG2 cells were inoculated with HEV virus samples and cultured with DMEM containing 2% calf serum for 4 days. The distribution of HEV in the cells was detected by immunofluorescence. PGC-A6 and miR-A6 inhibitor (anti-A6) were co transfected to A549, HepG2 cells were inoculated for 6 days, and cells were collected and collected. The effects of niR-A6 on the transcription of HEV ORF1 and ORF2mRNA were detected, and Western Blot technique was used to detect the regulation of miR-A6 on the expression of ORF2 protein in HEV. The study showed that miR-A6 had an enhanced effect on HEV both in mRNA level and protein level.
3 candidate target genes were screened in this paper. The interaction between candidate target gene and miR-A6 was detected by luciferase test. Real-time qPCR and Western Blot showed that miR-A6 may affect the replication of HEV by regulating the target gene. We think miR-A6 may pass the regulation of target gene and regulate the host's natural immune system. This study explored the mechanism of HEV replication and provided the basis for HEV vaccine research and antiviral drug research.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R373.21
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