戊型肝炎病毒編碼的microRNA-A6對(duì)病毒復(fù)制的影響
發(fā)布時(shí)間：2018-08-05 18:11
【摘要】：戊型肝炎病毒(Hepatitis E Virus, HEV)是一種經(jīng)糞口途徑傳播,嚴(yán)重危害人類健康的病毒性肝炎的病原體。HEV屬于肝炎病毒科,肝炎病毒屬,無(wú)囊膜的單股正鏈RNA病毒,由三個(gè)開放閱讀框(Open Reading Fragment, ORF)組成。HEV可以跨種間感染人和多種動(dòng)物。在發(fā)展中國(guó)家,戊型肝炎發(fā)病率較高,對(duì)孕婦危害尤為嚴(yán)重,妊娠晚期孕婦感染HEV后死亡率高達(dá)25%。 目前,HEV復(fù)制機(jī)制尚不清楚,microRNA (miR)是一類進(jìn)化上保守,在基因表達(dá)中起重要調(diào)控作用的小分子。病毒編碼的miR是一類潛在的、小的、非抗原性的基因表達(dá)調(diào)節(jié)因子,可以通過(guò)調(diào)節(jié)宿主細(xì)胞免疫系統(tǒng)來(lái)創(chuàng)造適宜病毒復(fù)制的環(huán)境。]microRNA-A6(miR-A6)是HEV編碼的其中一條miR,能夠增強(qiáng)HEV抗原在動(dòng)物體內(nèi)的表達(dá)。本試驗(yàn)旨在細(xì)胞水平研究miR-A6對(duì)HEV復(fù)制的影響,為研究HEV復(fù)制機(jī)制提供基礎(chǔ)。本研究工作包括3個(gè)部分： 1、miR-A6靶基因的篩選 利用TaregetScan5.1數(shù)據(jù)庫(kù)搜索,結(jié)合"microRNA-靶基因”共調(diào)節(jié)統(tǒng)計(jì)學(xué)分析法篩選得到候選靶基因：SIRP-α(signal-regulatory protein alpha)、ING5(inhibitor of growth family, member5)和ZAP (zinc-finger antiviral protein)。將上述候選靶基因的3’UTR插入到含熒光素酶(Luciferase)報(bào)告基因的pMIR-REPORT載體中,構(gòu)建候選靶基因報(bào)告載體。將三個(gè)候選靶基因報(bào)告載體分別和miR-A6類似物(miR-A6mimic)共轉(zhuǎn)染A549細(xì)胞,轉(zhuǎn)染24h后,熒光素酶實(shí)驗(yàn)證實(shí)miR-A6與SIRP-α、ING5和ZAP三個(gè)靶基因都具有相互作用。 2、miR-A6對(duì)候選靶基因的調(diào)控 構(gòu)建靶基因與綠色熒光蛋白(EGFP)報(bào)告基因融合表達(dá)載體,分別命名為pcDNA3.1(+)/SIRP-a-EGFP、pcDNA3.1(+)/ING5-EGFP、pcDNA3.1(+)/ZAP-EGFP,分別轉(zhuǎn)染HepG2細(xì)胞,通過(guò)觀察熒光證實(shí)所構(gòu)建的載體能在細(xì)胞中表達(dá)。miR-A6mimic分別與pcDNA3.1(+)/SIRP-α-EGFP、pcDNA3.1(+)/ING5-EGFP、pcDNA3.1(+)/ZAP-EGFP重組真核表達(dá)質(zhì)粒共轉(zhuǎn)染HEK293T細(xì)胞,利用Real-time qPCR技術(shù)檢測(cè)miR-A6對(duì)靶基因在mRNA水平的調(diào)控作用,利用Western Blot方法檢測(cè)miR-A6對(duì)靶基因在蛋白水平的調(diào)控作用。實(shí)驗(yàn)結(jié)果表明,miR-A6促進(jìn)靶基因ZAP表達(dá),抑制ING5和SIRP-α表達(dá)。 3、miR-A6對(duì)HEV復(fù)制的影響 MiR-A6mimic轉(zhuǎn)染A549、HepG2細(xì)胞24h后接種HEV病毒樣品,用含2%小牛血清的DMEM維持培養(yǎng)4天,通過(guò)免疫熒光方法檢測(cè)HEV在細(xì)胞中的分布；PGC-A6和miR-A6抑制物(anti-A6)共轉(zhuǎn)染A549、HepG2細(xì)胞24h后,接種HEV病毒樣品,維持培養(yǎng)6天,收集細(xì)胞,運(yùn)用Real-time qPCR方法檢測(cè)niR-A6對(duì)HEV ORF1和ORF2mRNA轉(zhuǎn)錄的調(diào)控作用；使用Western Blot技術(shù)檢測(cè)miR-A6對(duì)HEV ORF2蛋白表達(dá)的調(diào)控作用。研究表明,miR-A6對(duì)HEV無(wú)論在mRNA水平還是在蛋白水平都有增強(qiáng)作用。 本論文篩選得到3條候選靶基因,通過(guò)熒光素酶實(shí)驗(yàn)檢測(cè)了候選靶基因與miR-A6有相互作用,Real-time qPCR和Western Blot技術(shù)驗(yàn)證了miR-A6可能通過(guò)對(duì)靶基因的調(diào)控作用影響HEV的復(fù)制。我們認(rèn)為miR-A6可能通過(guò)調(diào)控靶基因,調(diào)節(jié)宿主天然免疫系統(tǒng)從而影響HEV的復(fù)制。本研究初步探索了HEV的復(fù)制機(jī)制,為HEV疫苗研究及抗病毒藥物研發(fā)提供基礎(chǔ)。
[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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R373.21
本文編號(hào)：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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R373.21
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相關(guān)期刊論文 前3條
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2 李寶安,王紅陽(yáng),陳正軍,吳孟超;信號(hào)調(diào)節(jié)蛋白SIRPα在肝癌內(nèi)的表達(dá)及意義[J];中華腫瘤雜志;1998年05期
3 ;Inhibition of hepatitis B virus gene expression and replication by artificial microRNA[J];World Journal of Gastroenterology;2008年29期
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