ORFV感染宿主細胞miRNA差異表達分析及VIR蛋白抗體制備與細胞定位觀察
發(fā)布時間：2018-07-13 15:07
【摘要】：羊傳染性膿皰病毒(orf virus,ORFV)引起的羊傳染性膿皰(contagious ecthyma)俗稱“羊口瘡”是一種重要的人獸共患性傳染病。羊口瘡的爆發(fā)和流行不但造成了嚴(yán)重的經(jīng)濟損失而且威脅人們身體健康。microRNA是一類長約22個核苷酸的非編碼單鏈RNA分子,主要作用于基因的轉(zhuǎn)錄后調(diào)控,在胚胎發(fā)育、細胞分化、細胞凋亡、造血功能和疾病發(fā)生等方面都具有重要的調(diào)控作用。ORFV的干擾素抗性基因(VIR基因)是其主要的毒力因子,對于病毒的復(fù)制具有重要作用。該基因作為ORFV的保守性基因,具有抗干擾素的功能,編碼感染早期的抗干擾素蛋白。本試驗以感染與非感染ORFV的羊皮膚成纖維細胞(GSFs)為研究對象,運用Solexa高通量測序技術(shù)對ORFV與GSFs互作過程中的micro RNA進行了探索與功能分析。結(jié)果表明,本實驗成功構(gòu)建了感染與非感染ORFV的GSFs細胞差異表達microRNA的表達譜,共獲得顯著上調(diào)microRNA共509個,顯著下調(diào)microRNA共169個,另有差異不顯著的microRNA共479個。micro RNA靶基因GO富集分析表明,在基因的分子功能顯著富集的條目中感染組GSFs細胞與非感染組GSFs細胞microRNA的主要差異在于感染組micro RNA主要在許多酶活性與核糖核苷酸的綁定活動中富集;在所處細胞位置顯著富集條目中感染組共7個主要與細胞膜、裂解泡、液泡和溶酶體相關(guān),而對照組只有2個主要與細胞膜相關(guān);在參與的生物過程顯著富集條目中感染組有2個主要與細胞脂質(zhì)代謝過程相關(guān),而感染組沒有分析出顯著富集的條目。通過micro RNA靶基因KEGG通路顯著性分析,并對感染組與非感染組富集的KEGG通路進行對比發(fā)現(xiàn):在ORFV與GSFs細胞互作過程中,主要使microRNA在細胞外基質(zhì)受體互作途徑、溶酶體、焦點粘連、白細胞遷移、Hedgehog信號通路、過氧化酶、膽汁分泌途徑、激動蛋白細胞骨架調(diào)節(jié)通路和視黃醇代謝通路中發(fā)生了富集。為了進一步研究ORFV的VIR蛋白的生物學(xué)特性,本試驗構(gòu)建了含有VIR基因的重組表達質(zhì)粒p ET-VIR和含有綠色熒光蛋白融合的真核重組質(zhì)粒p EGFP-VIR;免疫BALB/c小鼠制備了VIR蛋白的多克隆抗體。將質(zhì)粒pEGFP-VIR轉(zhuǎn)染綿羊睪丸細胞,觀察了VIR蛋白在細胞內(nèi)的表達分布情況。結(jié)果顯示,表達和純化了含有GST標(biāo)簽的GST-VIR蛋白,主要以可溶性形式存在,大小約27kDa,制備的多克隆抗體效價達1∶218,WB顯示抗體特異性較好,VIR蛋白主要分布于細胞核內(nèi)。本試驗通過分析感染與非感染ORFV的GSFs細胞差異表達microRNA的表達譜,共獲得顯著上調(diào)microRNA共509個,顯著下調(diào)microRNA共169個;在ORFV與GSFs細胞互作過程中,microRNA影響了許多酶的活性,參與核糖核苷酸的綁定活動和細胞脂質(zhì)代謝過程;microRNA可能在細胞外基質(zhì)受體互作、溶酶體活動、焦點粘連、白細胞遷移、過氧化酶活性、膽汁分泌途徑、激動蛋白細胞骨架調(diào)節(jié)和視黃醇代謝等通路中發(fā)揮了作用。本試驗還成功制備了VIR蛋白的多克隆抗體并探明了VIR蛋白主要在綿羊睪丸細胞的細胞核內(nèi)表達分布。
[Abstract]:Sheep infectious pustular virus (orf virus, ORFV) caused by infectious pustule (contagious ecthyma) commonly known as "sheep sore" is an important human zoonosis. The outbreak and epidemic of sheep mouth ulcer not only cause serious economic loss but also threaten people's health.MicroRNA is a class of 22 nucleotides non coding single. Chain RNA molecules, which are mainly responsible for post transcriptional regulation of genes, play an important regulatory role in embryonic development, cell differentiation, cell apoptosis, hematopoietic function and disease occurrence. The.ORFV interferon resistance gene (VIR gene) is its main virulence factor and plays an important role in the replication of the disease. This gene is conserved as a ORFV. Sex genes, which have the function of anti interferon, encode anti interferon protein in early infection. In this experiment, the sheep skin fibroblasts (GSFs) infected with non infected ORFV (GSFs) were used as the research object. The micro RNA in the interaction between ORFV and GSFs was explored and functional analyzed by Solexa high throughput sequencing. The results showed that the experiment was successfully constructed. The expression profiles of differential expression of microRNA in GSFs cells from infected and non infected ORFV were built, and a total of 509 microRNA were significantly up-regulated, and 169 microRNA were significantly down, and there were no significant differences in microRNA and 479.Micro RNA target genes GO enrichment analysis. The main difference in the microRNA of GSFs cells in the infection group was that the micro RNA in the infection group was enriched mainly in the binding activities of a number of enzyme activities and ribonucleotide, and 7 of the infected groups were mainly related to cell membrane, lysis vesicles, vacuoles and lysosomes, while only 2 of the control groups were mainly related to the cell membrane. In the significant enrichment items of the biological process, 2 mainly related to the lipid metabolism in the cell, but the infection group did not analyze the significant enrichment items. The significance of the KEGG pathway of the micro RNA target gene was analyzed and the KEGG pathway enriched with the non infected group was compared and found in the interaction of ORFV and GSFs cells, The main results are that microRNA has been enriched in the intercellular matrix receptor interaction pathway, lysosome, focal adhesion, leukocyte migration, Hedgehog signaling pathway, peroxidase, bile secretion pathway, agonist cytoskeleton regulation pathway and retinol metabolic pathway. In order to further study the biological characteristics of ORFV VIR protein, this experiment was constructed. The recombinant expression plasmid P ET-VIR containing the VIR gene and the eukaryotic recombinant plasmid P EGFP-VIR containing the fusion of green fluorescent protein, and the immunized BALB/c mice were prepared for the polyclonal antibody of the VIR protein. The plasmid pEGFP-VIR was transfected into the sheep testicular cells and the expression and distribution of VIR protein in the cells was observed. The results showed that the expression and purification of the expression and purification of GST were contained in the expression and purification of GST. The GST-VIR protein of the label, mainly in the soluble form, was about 27kDa, the potency of the polyclonal antibody was 1: 218, the WB showed that the antibody specificity was good, and the VIR protein was mainly distributed in the nucleus. By analyzing the expression profiles of microRNA in the GSFs cells of the infected and non infected ORFV, the total number of microRNA was up to be up to be up to 5. 09, a total of 169 down-regulation of microRNA; in the interaction between ORFV and GSFs cells, microRNA affects the activity of many enzymes, participates in the binding activity of ribonucleotide and the process of cell lipid metabolism; microRNA may be interacted on the extracellular matrix receptor, lysosome activity, focal adhesion, leukocyte migration, peroxidase activity, and bile secretion pathway In this experiment, the polyclonal antibody of VIR protein and the expression of VIR protein mainly in the nucleus of sheep testis cells were also successfully prepared.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
本文編號：2119816
[Abstract]:Sheep infectious pustular virus (orf virus, ORFV) caused by infectious pustule (contagious ecthyma) commonly known as "sheep sore" is an important human zoonosis. The outbreak and epidemic of sheep mouth ulcer not only cause serious economic loss but also threaten people's health.MicroRNA is a class of 22 nucleotides non coding single. Chain RNA molecules, which are mainly responsible for post transcriptional regulation of genes, play an important regulatory role in embryonic development, cell differentiation, cell apoptosis, hematopoietic function and disease occurrence. The.ORFV interferon resistance gene (VIR gene) is its main virulence factor and plays an important role in the replication of the disease. This gene is conserved as a ORFV. Sex genes, which have the function of anti interferon, encode anti interferon protein in early infection. In this experiment, the sheep skin fibroblasts (GSFs) infected with non infected ORFV (GSFs) were used as the research object. The micro RNA in the interaction between ORFV and GSFs was explored and functional analyzed by Solexa high throughput sequencing. The results showed that the experiment was successfully constructed. The expression profiles of differential expression of microRNA in GSFs cells from infected and non infected ORFV were built, and a total of 509 microRNA were significantly up-regulated, and 169 microRNA were significantly down, and there were no significant differences in microRNA and 479.Micro RNA target genes GO enrichment analysis. The main difference in the microRNA of GSFs cells in the infection group was that the micro RNA in the infection group was enriched mainly in the binding activities of a number of enzyme activities and ribonucleotide, and 7 of the infected groups were mainly related to cell membrane, lysis vesicles, vacuoles and lysosomes, while only 2 of the control groups were mainly related to the cell membrane. In the significant enrichment items of the biological process, 2 mainly related to the lipid metabolism in the cell, but the infection group did not analyze the significant enrichment items. The significance of the KEGG pathway of the micro RNA target gene was analyzed and the KEGG pathway enriched with the non infected group was compared and found in the interaction of ORFV and GSFs cells, The main results are that microRNA has been enriched in the intercellular matrix receptor interaction pathway, lysosome, focal adhesion, leukocyte migration, Hedgehog signaling pathway, peroxidase, bile secretion pathway, agonist cytoskeleton regulation pathway and retinol metabolic pathway. In order to further study the biological characteristics of ORFV VIR protein, this experiment was constructed. The recombinant expression plasmid P ET-VIR containing the VIR gene and the eukaryotic recombinant plasmid P EGFP-VIR containing the fusion of green fluorescent protein, and the immunized BALB/c mice were prepared for the polyclonal antibody of the VIR protein. The plasmid pEGFP-VIR was transfected into the sheep testicular cells and the expression and distribution of VIR protein in the cells was observed. The results showed that the expression and purification of the expression and purification of GST were contained in the expression and purification of GST. The GST-VIR protein of the label, mainly in the soluble form, was about 27kDa, the potency of the polyclonal antibody was 1: 218, the WB showed that the antibody specificity was good, and the VIR protein was mainly distributed in the nucleus. By analyzing the expression profiles of microRNA in the GSFs cells of the infected and non infected ORFV, the total number of microRNA was up to be up to be up to 5. 09, a total of 169 down-regulation of microRNA; in the interaction between ORFV and GSFs cells, microRNA affects the activity of many enzymes, participates in the binding activity of ribonucleotide and the process of cell lipid metabolism; microRNA may be interacted on the extracellular matrix receptor, lysosome activity, focal adhesion, leukocyte migration, peroxidase activity, and bile secretion pathway In this experiment, the polyclonal antibody of VIR protein and the expression of VIR protein mainly in the nucleus of sheep testis cells were also successfully prepared.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
【共引文獻】
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1 王國慶;朱紫祥;曹偉軍;劉磊;鄭海學(xué);;RNA病毒阻斷RIG-I樣受體識別dsRNA機制研究進展[J];病毒學(xué)報;2014年06期
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3 譚強;羊口瘡病毒的分離鑒定及B2L囊膜蛋白單克隆抗體的制備[D];黑龍江八一農(nóng)墾大學(xué);2014年
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8 張瑜;羊口瘡病毒楊凌株和瀘西株的分離鑒定及毒力致弱研究[D];西北農(nóng)林科技大學(xué);2014年
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10 尚辰;陜西關(guān)中某羊場羊口瘡病毒的分離鑒定與基因序列分析[D];西北農(nóng)林科技大學(xué);2014年
，本文編號：2119816
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