基于转录组测序的绵羊MBL抗支原体肺炎相关基因的筛选与分析

发布时间：2018-02-14 10:37

本文关键词： 转录组高通量测序绵羊肺炎支原体绵羊呼吸道上皮细胞甘露糖结合凝集素　出处：《石河子大学》2017年硕士论文　论文类型：学位论文

【摘要】：绵羊支原体肺炎(Mycoplasma pneumonia,MP)在新疆乃至世界范围内威胁着养羊业的发展,是由绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)感染引起的慢性呼吸道传染病。该病的致病机理比较复杂且尚不明确,因此诊断、治疗和预防的难度较大。甘露聚糖结合凝集素(Mannan-binding lectin,MBL)是机体天然免疫系统中重要的组成,在绵羊抗MO感染中发挥着一定作用。目的:为了探讨MO的致病机制和MBL在机体抗MO感染中发挥的抗病机制,从转录组测序结果中筛选差异表达基因,并完成部分差异基因在细胞水平的验证。方法:(1)通过真核表达和血清提取分别获取绵羊MBL重组蛋白与天然蛋白,将2月龄左右中国美利奴羊通过PCR-SSCP将绵羊MBL外显子1基因分型,根据分型结果,随机选取抗病型绵羊5只与易感型绵羊5只,进行MO人工感染。感染后14d、21d静脉注射MBL,42d屠宰取肝脏送交生物公司测序。借助生物信息学分析软件对测序反馈结果进行数据分析,筛选出差异表达基因。(2)组织块法培养绵羊呼吸道上皮细胞,分离纯化出绵羊呼吸道上皮细胞,并进行形态学与免疫组化鉴定(3)培养绵羊呼吸道上皮细胞,于MO感染8h、16h、24h检测细胞存活率。荧光定量PCR检测FGF1、C-Myc、c-jun基因在MO感染24h的转录水平。结果:(1)SDS-PAGE凝胶可在28kD及更高分子量处检测到天然MBL单体和聚合体,重组蛋白仅在约32kD处呈现单一的清晰条带。单糖配体结合试验显示二者都具有较好的生物活性;PCR-SSCP分选出中国美利奴羊4种MBL外显子1基因型;肝脏取样测序成功构建MO感染、MBL治疗模型的转录组文库。对转录组文库进行数据分析,成功筛选出差异表达显著的基因325个,并对其进行了归类与转录本表达水平等分析。(2)成功从绵羊气管分离纯化得到绵羊呼吸道上皮细胞与成纤维细胞,并对绵羊呼吸道上皮细胞完成了免疫组化鉴定。(3)易感组与抗病组绵羊呼吸道上皮细胞在MO感染8h、16h、24h时的细胞存活率不断下降,但易感组与抗病组之间无显著性差异(P0.05)。细胞培养基中加入MBL可使MO侵染24h导致的细胞凋亡率下降,差异显著(P0.05)。MO感染24h后FGF1基因转录水平比未经感染的细胞显著性升高(P0.01);C-Myc、c-jun基因在MO感染24h后与未经感染细胞相比无显著性差异(P0.05)。结论:(1)通过高通量测序成功构建了中国美利奴羊人工感染MO且经MBL治疗的转录组文库,对文库分析获得325个差异表达基因。(2)成功分离出绵羊呼吸道上皮细胞与成纤维细胞。(3)不同基因型绵羊的细胞在抗MO感染未表现出差异;绵羊呼吸道上皮细胞经MO感染,FGF1基因转录显著性升高,c-jun、C-Myc基因转录无显著变化。MBL在细胞水平及个体水平对绵羊抗肺炎支原体感染均具有一定保护作用,MBL对细胞凋亡的保护机制中,FGF1可能发挥着一定作用。
[Abstract]:Mycoplasma pneumoniae MPN (Mycoplasma pneumoniae), which threatens the development of sheep industry in Xinjiang and the world, is a chronic respiratory infectious disease caused by the infection of Mycoplasma ovipneumoniae (MOA) in sheep. The pathogenesis of Mycoplasma pneumoniae is complex and unclear, so the diagnosis of the disease is not clear. Mannan-binding lectin (MBL) is an important component of the innate immune system. Objective: to investigate the pathogenesis of MO and the resistance of MBL to MO infection in sheep. Methods the recombinant protein and natural protein of sheep MBL were obtained by eukaryotic expression and serum extraction, respectively. Chinese Merino sheep at about 2 months of age were genotyped into sheep MBL exon 1 by PCR-SSCP. According to the typing results, 5 resistant sheep and 5 susceptible sheep were randomly selected. MO artificial infection was performed. The liver was slaughtered and sent to the biological company for sequencing at 14 days and 21 days after the infection. The data of the feedback results were analyzed by bioinformatics analysis software. Sheep respiratory epithelial cells were isolated and purified by tissue mass method, and identified by morphological and immunohistochemical methods. The survival rate of the cells was detected at 8 h, 16 h and 24 h after MO infection. The transcription level of FGF1C Mycfon c-jun gene was detected by fluorescence quantitative PCR at 24 h after MO infection. Results the natural MBL monomer and polymer could be detected at 28kD and higher molecular weight by the SDS-PAGE gel. The recombinant protein only showed a clear band at about 32kD. The results of monosaccharide ligand binding test showed that both of them had good bioactivity and PCR-SSCP, and 4 MBL exon 1 genotypes were selected from Chinese Merino sheep. The transcriptional library of MO infected MBL model was successfully constructed by liver sampling and sequencing. 325 differentially expressed genes were successfully screened by data analysis of the transcriptional library. The epithelial cells and fibroblasts of sheep respiratory tract were isolated and purified from sheep trachea. The survival rate of sheep respiratory epithelial cells in susceptible and disease-resistant groups decreased continuously after MO infection for 8 h or 16 h or 24 h. However, there was no significant difference between susceptible group and disease resistant group (P 0.05). Adding MBL to cell culture medium could decrease the apoptosis rate induced by MO infection for 24 h. The transcription level of FGF1 gene was significantly higher than that of uninfected cells 24 hours after infection. There was no significant difference in the transcription level of P0.01C ~ (c) c-jun gene between infected and uninfected cells 24 hours after MO infection. Conclusion: 1) A high throughput sequencing method was used to construct the gene. Transcriptome library of Homelino sheep artificially infected with MO and treated with MBL. A total of 325 differentially expressed genes were obtained by library analysis.) the sheep respiratory epithelial cells and fibroblasts were isolated successfully. The cells of different genotypes of sheep showed no difference in resistance to MO infection. Significant increase of FGF1 Gene transcription in Sheep Respiratory tract epithelial cells with MO infection. There is no significant change in c-junn C-Myc transcription.MBL has a protective effect on mycoplasma pneumoniae infection in sheep at cell level and individual level; MBL can protect against mycoplasma pneumoniae infection in sheep. FGF1 may play a role in the protective mechanism of FGF1.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.26

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