ORFV感染宿主细胞miRNA差异表达分析及VIR蛋白抗体制备与细胞定位观察
发布时间：2018-07-13 15:07
【摘要】：羊传染性脓疱病毒(orf virus,ORFV)引起的羊传染性脓疱(contagious ecthyma)俗称“羊口疮”是一种重要的人兽共患性传染病。羊口疮的爆发和流行不但造成了严重的经济损失而且威胁人们身体健康。microRNA是一类长约22个核苷酸的非编码单链RNA分子,主要作用于基因的转录后调控,在胚胎发育、细胞分化、细胞凋亡、造血功能和疾病发生等方面都具有重要的调控作用。ORFV的干扰素抗性基因(VIR基因)是其主要的毒力因子,对于病毒的复制具有重要作用。该基因作为ORFV的保守性基因,具有抗干扰素的功能,编码感染早期的抗干扰素蛋白。本试验以感染与非感染ORFV的羊皮肤成纤维细胞(GSFs)为研究对象,运用Solexa高通量测序技术对ORFV与GSFs互作过程中的micro RNA进行了探索与功能分析。结果表明,本实验成功构建了感染与非感染ORFV的GSFs细胞差异表达microRNA的表达谱,共获得显著上调microRNA共509个,显著下调microRNA共169个,另有差异不显著的microRNA共479个。micro RNA靶基因GO富集分析表明,在基因的分子功能显著富集的条目中感染组GSFs细胞与非感染组GSFs细胞microRNA的主要差异在于感染组micro RNA主要在许多酶活性与核糖核苷酸的绑定活动中富集;在所处细胞位置显著富集条目中感染组共7个主要与细胞膜、裂解泡、液泡和溶酶体相关,而对照组只有2个主要与细胞膜相关;在参与的生物过程显著富集条目中感染组有2个主要与细胞脂质代谢过程相关,而感染组没有分析出显著富集的条目。通过micro RNA靶基因KEGG通路显著性分析,并对感染组与非感染组富集的KEGG通路进行对比发现:在ORFV与GSFs细胞互作过程中,主要使microRNA在细胞外基质受体互作途径、溶酶体、焦点粘连、白细胞迁移、Hedgehog信号通路、过氧化酶、胆汁分泌途径、激动蛋白细胞骨架调节通路和视黄醇代谢通路中发生了富集。为了进一步研究ORFV的VIR蛋白的生物学特性,本试验构建了含有VIR基因的重组表达质粒p ET-VIR和含有绿色荧光蛋白融合的真核重组质粒p EGFP-VIR;免疫BALB/c小鼠制备了VIR蛋白的多克隆抗体。将质粒pEGFP-VIR转染绵羊睾丸细胞,观察了VIR蛋白在细胞内的表达分布情况。结果显示,表达和纯化了含有GST标签的GST-VIR蛋白,主要以可溶性形式存在,大小约27kDa,制备的多克隆抗体效价达1∶218,WB显示抗体特异性较好,VIR蛋白主要分布于细胞核内。本试验通过分析感染与非感染ORFV的GSFs细胞差异表达microRNA的表达谱,共获得显著上调microRNA共509个,显著下调microRNA共169个;在ORFV与GSFs细胞互作过程中,microRNA影响了许多酶的活性,参与核糖核苷酸的绑定活动和细胞脂质代谢过程;microRNA可能在细胞外基质受体互作、溶酶体活动、焦点粘连、白细胞迁移、过氧化酶活性、胆汁分泌途径、激动蛋白细胞骨架调节和视黄醇代谢等通路中发挥了作用。本试验还成功制备了VIR蛋白的多克隆抗体并探明了VIR蛋白主要在绵羊睾丸细胞的细胞核内表达分布。
[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.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：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.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
【共引文献】
相关期刊论文 前2条
1 王国庆;朱紫祥;曹伟军;刘磊;郑海学;;RNA病毒阻断RIG-I样受体识别dsRNA机制研究进展[J];病毒学报;2014年06期
2 刘其昌;宋德荣;彭华;周大荣;张琼娣;罗耀;郭振刚;马金萍;;羊口疮研究进展[J];当代畜牧;2015年18期
相关博士学位论文 前2条
1 邵洪泽;羊传染性脓疱病毒FIL基因克隆表达及分子生物学检测方法的建立与初步应用[D];吉林农业大学;2013年
2 李吉达;宿主细胞CyPB在Orf病毒感染过程中功能的初步研究[D];吉林大学;2014年
相关硕士学位论文 前10条
1 李前瑞;羊口疮病毒PCR检测方法的建立与应用[D];西北农林科技大学;2013年
2 李杰;羊口疮抗体ELISA检测方法的建立及应用[D];西北农林科技大学;2013年
3 谭强;羊口疮病毒的分离鉴定及B2L囊膜蛋白单克隆抗体的制备[D];黑龙江八一农垦大学;2014年
4 包英夫;抗羊传染性脓疱病毒免疫球蛋白可变区多样性研究[D];吉林大学;2014年
5 孔汉金;羊口疮病毒感染宿主细胞的蛋白质组变化及功能分析[D];华中农业大学;2014年
6 郑泽微;羊口疮病毒编码的一种新型NF-κB核抑制剂002蛋白的分子机制[D];南方医科大学;2014年
7 汪园园;羊口疮病毒编码的024蛋白调控NF-κB信号通路的分子机制[D];南方医科大学;2014年
8 张瑜;羊口疮病毒杨凌株和泸西株的分离鉴定及毒力致弱研究[D];西北农林科技大学;2014年
9 高晶晖;羊口疮病毒威海株和昆明株的分离鉴定及毒力致弱研究[D];西北农林科技大学;2014年
10 尚辰;陕西关中某羊场羊口疮病毒的分离鉴定与基因序列分析[D];西北农林科技大学;2014年
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