本文關(guān)鍵詞： 轉(zhuǎn)錄組 高通量測(cè)序 綿羊肺炎支原體 綿羊呼吸道上皮細(xì)胞 甘露糖結(jié)合凝集素　出處：《石河子大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：綿羊支原體肺炎(Mycoplasma pneumonia,MP)在新疆乃至世界范圍內(nèi)威脅著養(yǎng)羊業(yè)的發(fā)展,是由綿羊肺炎支原體(Mycoplasma ovipneumoniae,MO)感染引起的慢性呼吸道傳染病。該病的致病機(jī)理比較復(fù)雜且尚不明確,因此診斷、治療和預(yù)防的難度較大。甘露聚糖結(jié)合凝集素(Mannan-binding lectin,MBL)是機(jī)體天然免疫系統(tǒng)中重要的組成,在綿羊抗MO感染中發(fā)揮著一定作用。目的:為了探討MO的致病機(jī)制和MBL在機(jī)體抗MO感染中發(fā)揮的抗病機(jī)制,從轉(zhuǎn)錄組測(cè)序結(jié)果中篩選差異表達(dá)基因,并完成部分差異基因在細(xì)胞水平的驗(yàn)證。方法:(1)通過(guò)真核表達(dá)和血清提取分別獲取綿羊MBL重組蛋白與天然蛋白,將2月齡左右中國(guó)美利奴羊通過(guò)PCR-SSCP將綿羊MBL外顯子1基因分型,根據(jù)分型結(jié)果,隨機(jī)選取抗病型綿羊5只與易感型綿羊5只,進(jìn)行MO人工感染。感染后14d、21d靜脈注射MBL,42d屠宰取肝臟送交生物公司測(cè)序。借助生物信息學(xué)分析軟件對(duì)測(cè)序反饋結(jié)果進(jìn)行數(shù)據(jù)分析,篩選出差異表達(dá)基因。(2)組織塊法培養(yǎng)綿羊呼吸道上皮細(xì)胞,分離純化出綿羊呼吸道上皮細(xì)胞,并進(jìn)行形態(tài)學(xué)與免疫組化鑒定(3)培養(yǎng)綿羊呼吸道上皮細(xì)胞,于MO感染8h、16h、24h檢測(cè)細(xì)胞存活率。熒光定量PCR檢測(cè)FGF1、C-Myc、c-jun基因在MO感染24h的轉(zhuǎn)錄水平。結(jié)果:(1)SDS-PAGE凝膠可在28kD及更高分子量處檢測(cè)到天然MBL單體和聚合體,重組蛋白僅在約32kD處呈現(xiàn)單一的清晰條帶。單糖配體結(jié)合試驗(yàn)顯示二者都具有較好的生物活性;PCR-SSCP分選出中國(guó)美利奴羊4種MBL外顯子1基因型;肝臟取樣測(cè)序成功構(gòu)建MO感染、MBL治療模型的轉(zhuǎn)錄組文庫(kù)。對(duì)轉(zhuǎn)錄組文庫(kù)進(jìn)行數(shù)據(jù)分析,成功篩選出差異表達(dá)顯著的基因325個(gè),并對(duì)其進(jìn)行了歸類與轉(zhuǎn)錄本表達(dá)水平等分析。(2)成功從綿羊氣管分離純化得到綿羊呼吸道上皮細(xì)胞與成纖維細(xì)胞,并對(duì)綿羊呼吸道上皮細(xì)胞完成了免疫組化鑒定。(3)易感組與抗病組綿羊呼吸道上皮細(xì)胞在MO感染8h、16h、24h時(shí)的細(xì)胞存活率不斷下降,但易感組與抗病組之間無(wú)顯著性差異(P0.05)。細(xì)胞培養(yǎng)基中加入MBL可使MO侵染24h導(dǎo)致的細(xì)胞凋亡率下降,差異顯著(P0.05)。MO感染24h后FGF1基因轉(zhuǎn)錄水平比未經(jīng)感染的細(xì)胞顯著性升高(P0.01);C-Myc、c-jun基因在MO感染24h后與未經(jīng)感染細(xì)胞相比無(wú)顯著性差異(P0.05)。結(jié)論:(1)通過(guò)高通量測(cè)序成功構(gòu)建了中國(guó)美利奴羊人工感染MO且經(jīng)MBL治療的轉(zhuǎn)錄組文庫(kù),對(duì)文庫(kù)分析獲得325個(gè)差異表達(dá)基因。(2)成功分離出綿羊呼吸道上皮細(xì)胞與成纖維細(xì)胞。(3)不同基因型綿羊的細(xì)胞在抗MO感染未表現(xiàn)出差異;綿羊呼吸道上皮細(xì)胞經(jīng)MO感染,FGF1基因轉(zhuǎn)錄顯著性升高,c-jun、C-Myc基因轉(zhuǎn)錄無(wú)顯著變化。MBL在細(xì)胞水平及個(gè)體水平對(duì)綿羊抗肺炎支原體感染均具有一定保護(hù)作用,MBL對(duì)細(xì)胞凋亡的保護(hù)機(jī)制中,FGF1可能發(fā)揮著一定作用。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.26

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