本文關鍵詞： PRRSV PAM microRNAs 高通量測序 通城豬　出處：《華中農業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：豬繁殖與呼吸綜合征(Porcine Reproductive and Respiratory Syndrome,PRRS)俗稱藍耳病,以流行地域廣、傳播速度快、感染率高等特點成為全球規(guī)模化豬場的主要疫病之一,主要造成妊娠母豬的繁殖障礙及引起各種年齡豬特別是仔豬的呼吸道疾病。PRRS是由豬繁殖與呼吸綜合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)引起的,PRRSV是一種單股正鏈RNA病毒,具有易變性且能引起宿主的免疫抑制,給獸醫(yī)臨床進行該病的控制帶來很大困難,目前尚無有效的防控辦法。通城豬是我國優(yōu)良的地方品種,具有抗逆性強,肉質鮮嫩等特點,在HP-PRRS爆發(fā)期間,地處湖北省通城縣境內的通城豬卻無一例是因感染HP-PRRS而死亡的報道,故推測通城豬對HP-PRRS具有一定的抗性。Micro RNA(mi RNA)作為一類由內源基因編碼的長度約為20-24個核苷酸的非編碼單鏈RNA分子,在原核生物到哺乳動物中廣泛分布并普遍參與機體內多種生命過程的調控,已有報道m(xù)i R-181、mi R-125b等能抑制PRRSV在宿主中的增殖,在PRRSV感染機體后是否有大量的mi RNAs參與抗病毒作用尚且不知。本研究以通城豬和大白豬為材料,利用人工感染PRRSV后和感染前的肺泡巨噬細胞(Porcine Alveolar Macrophages,PAMs)進行小RNA高通量測序,篩選在兩品種感染前后和品種間PAMs中差異表達的mi RNAs并分析其調控網絡,為進一步篩選通城豬中的抗PRRSV的mi RNAs奠定基礎。本研究的主要內容及結果如下:(1)人工感染PRRSV后,通城豬的肺部病變和炎癥比大白豬輕,肺組織中的病毒載量低于大白豬。利用5周齡且PRRSV、PCV、PRV三種病毒都為陰性的健康斷奶仔豬通城豬22頭和大白豬12頭,按照同胞關系均勻隨機分為感染組和對照組,經過預試驗后進行人工感染,試驗組豬肌肉注射PRRSV-Wu H3毒株,對照組豬肌肉注射DMEM;感染后第7天剖檢,取肺臟等組織器官,每個肺臟的一側采用PBS經支氣管肺泡灌洗技術來收集PAMs,提取總RNA后用于小RNA的高通量測序,另一側采集肺組織樣品后用4%的多聚甲醛固定,用來制作病理切片并進行免疫組化分析。臨床觀察發(fā)現,6頭感染組豬在2-5天內先后出現精神沉郁,采食下降、嘔吐、喘氣、高溫、發(fā)熱等癥狀,感染組的大白豬比通城豬體溫升高更多,病癥更明顯;解剖時觀察肺部大體病變,發(fā)現感染組的大白豬肺臟明顯水腫,表面有大量出血點,肺葉出現深紅色實變區(qū),淋巴結廣泛腫大,通城豬肺部腫脹,實變程度比大白豬輕,粉色有彈性,呈海綿狀。用采集的肺組織樣制作石蠟切片,經HE染色后于倒置顯微鏡下觀察,發(fā)現通城豬和大白豬感染PRRSV后,肺臟均出現間質性肺炎的病變,細支氣管和血管周圍有單核巨噬細胞浸潤,大部分肺泡壁顯著增厚,肺泡腔狹窄,腔內主要可見壞死細胞,有均質紅染的漿液及巨噬細胞,從組織學病變的嚴重程度比較,大白豬較通城豬更嚴重。對照組的通城豬和大白豬肺臟組織結構清楚,未見異常變化。用辣根過氧化物酶標記的PRRSV山羊抗鼠Ig G抗體通過免疫組化法來觀察PRRSV抗原在肺臟中的分布,在顯微鏡完全相同的圖像采集條件下,每張切片在40倍鏡下隨機選取至少5個視野,用軟件IPP6.0進行定量分析,觀察發(fā)現PRRSV抗體陽性反應產物在感染組豬肺臟中主要分布于肺泡巨噬細胞、肺泡II型上皮細胞、淋巴細胞及脫落細胞,在同一品種內,相對于對照組而言,感染組的豬肺臟中PRRSV陽性信號明顯分布,而在感染組內,通城豬肺臟中PRRSV的分布量少于大白豬。(2)通城豬和大白豬感染PRRSV前后的12個PAM樣本進行小RNA組的高通量測序,每個樣本得到至少0.5G的數據,獲得至少10327597條原始序列,比對得到391個已知豬的成熟mi RNA,其中67個在不同組合中差異表達。選擇12頭豬的PAM作為測序樣本,提取總RNA后構建12個c DNA文庫,于Illumina Hi Seq2000平臺進行小RNA組的高通量測序,每個樣本得到的原始數據量都在0.5G以上,至少獲得10327597條原始序列,過濾后得到的純凈序列reads數均占原始序列總reads數的95%以上,20bp左右的堿基錯誤率都在0.02%以內。利用mi RDeep2將測序序列與Gene Bank中豬基因組序列及mi RBase中成熟mi RNA序列進行比對,發(fā)現在12個樣本中均得到391個已報道的豬的成熟mi RNA。兩兩組合T檢驗篩選出了67個差異表達mi RNAs,篩選標準為T-test的P值≤0.05,fold change1為上調,fold change1為下調,其中通城豬感染組與對照組(TC-INJ-CON)相比顯著下調表達的mi RNA有4個(mi R-101、mi R-146b、mi R-296-3p和mi R-664-5p),而上調表達的mi RNA有5個(mi R-22-3p、mi R-24-2-5p、mi R-27b-5p、mi R-423-5p和mi R-7137-5p);而大白豬感染組與對照組(LW-INJ-CON)相比顯著下調表達的mi RNA有36個,上調表達的mi RNA有20個;通城豬感染組與大白豬感染組(TC-INF-LW)相比,有5個下調的mi RNA和3個上調的mi RNA;通城豬對照組和大白豬對照組(TC-CON-LW)比較,有4個下調mi RNA和3個上調mi RNA。其中,12個mi RNAs出現在多個組合中,例如mi R-101在TC-INF-CON中下調,而在LW-INF-CON和TC-CON-LW中上調,mi R-296-3p和mi R146b在TC-INF-CON和LW-INF-CON兩個組合中均下調,而mi R-423-5p和mi R7137-5p在TC-INF-CON和LW-INF-CON兩個組合都是上調。(3)預測到67個差異表達mi RNAs的宿主靶基因有22234個,在PRRSV基因組上的結合靶點有119個,宿主靶基因富集到多條GO Terms和KEGG通路中,并形成mi RNA-m RNA調控網絡。利用軟件Target Scan Human6.2預測差異表達mi RNAs在豬基因組中的靶基因,得到22243個m RNA-mi RNA互作位點,利用軟件mi Randa預測到差異表達mi RNA在PRRSV基因組上有119個結合靶點。通城豬感染組與對照組差異表達mi RNAs的靶基有4474個,富集到801條GO Terms和50個KEGG通路中,大白豬感染組與對照組差異表達mi RNAs的靶基有12856個,富集到901條GO Terms和57個KEGG通路中。在二者共有的42個通路中,有12個與信號轉導相關的通路和18個與疾病癌癥相關的通路,還有巨噬細胞內受體FcγR介導的吞噬作用通路,推測此通路可能與宿主抗病毒的先天性免疫或PRRSV侵入機制有關。結合本課題組轉錄組測序數據的分析結果(另文見博士生梁婉的論文),將通城豬的差異表達mi RNA和m RNA聯合分析,發(fā)現有8個差異表達mi RNAs能靶向143個差異表達基因,其中4個下調的mi RNA對應37個上調的基因,4個上調的mi RNA對應38個下調的基因,將143個差異基因用在線軟件DAVID作GO和KEGG分析,富集到51個生物學過程(BP)、17種細胞組分(CC)和10種生物學功能(MF)的分類中,參與到p53信號通路、JAK-STAT信號通路、癌癥通路、細胞因子及其受體的相互作用中,利用Cytoscape軟件制作mi RNA——m RNA的蛋白調控關系網絡,發(fā)現mi R-101和mi R-25b-5p等對多個靶基都具有調控作用。
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) commonly known as blue ear disease, in broad areas, spread fast, high rate of infection has become one of the main diseases of global scale pig farms, mainly caused by reproductive failure of pregnant sows and pigs of all ages, especially in piglets caused by respiratory disease caused by porcine.PRRS reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV) caused by PRRSV, is a kind of positive single strand RNA virus, which is changeable and can cause host immune suppression, control to the veterinary clinic for the disease has caused great difficulties, there is no effective prevention and control measures is Tongcheng. China's fine local variety, with strong resistance, fresh meat and other characteristics, during the HP-PRRS outbreak, the territory is located in Tongcheng County of Hubei Province Tongcheng but no example is due HP-PRRS infection and death have been reported, suggesting that Tongcheng resistant.Micro RNA on HP-PRRS (MI RNA) is a kind of endogenous gene encoding by the length of single stranded RNA molecules encoding non 20-24 nucleotides, in the regulation of prokaryotes to mammals are widely distributed and widely participate in a variety of life process in the body, have been reported mi R-181, MI R-125b can inhibit PRRSV proliferation in the host, whether in the PRRSV infection of a large number of MI RNAs is involved in the antiviral effect unknown. In this study, Tongcheng and large white pigs as materials, using human alveolar macrophages after PRRSV infection and infection before (Porcine Alveolar Macrophages, PAMs) are small RNA high-throughput sequencing, screening in two varieties of PAMs infection in MI and Varietal Difference before and after RNAs and analysis of the regulatory network for further screening of Tongcheng pigs against PRRSV mi R NAs foundation. The main contents and results of this study are as follows: (1) PRRSV after artificial infection, Tongcheng lung lesions and inflammation than large white light load in lung virus below Yorkshire. By 5 weeks of age and PRRSV, PCV, PRV three kinds of virus were negative healthy weaned piglets Tongcheng pigs 22 large white and 12 head, according to the uniform sibling were randomly divided into infection group and control group, after the pre test after artificial infection test group porcine intramuscular injection of PRRSV-Wu H3 strains, the control group pigs by intramuscular injection of DMEM infection; seventh days after the autopsy, the lungs and other organs, each side of the lung using PBS technology to collect by bronchoalveolar lavage PAMs, the total RNA was extracted for high-throughput sequencing of small RNA, the other side to collect lung tissue samples were fixed with 4% paraformaldehyde, used to make pathological and immunohistochemical analysis. Clinical observation, 6 The pig infection group in 2-5 days has appeared in depression, vomiting, decreased food intake, breathing, temperature, fever and other symptoms, the infection group increased more than the large white pigs of body temperature, symptoms are more obvious; anatomical observation of pulmonary pathological changes, found that the infection group significantly large white lung edema, surface bleeding deep red, lobar consolidation, extensive lymph node enlargement, Tongcheng pig lungs swelling and consolidation degree than the large white light pink, elastic, spongy. With the acquisition of the lung tissue samples in paraffin, stained by HE in inverted microscope observation, found in Tongcheng and large white pigs infected after PRRSV, the lungs showed interstitial pneumonia lesions around the bronchioles and vascular infiltration of mononuclear macrophage, most of alveolar wall thickening of alveolar cavity stenosis, mainly necrotic cells, and macrophages have serous homogeneous red staining, from Comparison of the severity of histological lesions, a large white Tongcheng more serious. The control group of Tongcheng and large white lung tissue structure is clear, no abnormal changes. Using horseradish peroxidase labeled Goat anti mouse G antibody PRRSV Ig to observe the distribution of PRRSV antigen in the lung by immunohistochemical method, image acquisition conditions is the same in the microscope, each slice in 40 magnification were randomly selected at least 5 horizons were quantitatively analyzed by IPP6.0 software, PRRSV antibody positive reaction in pig lung infection group distributed mainly in alveolar macrophages was observed, alveolar type II epithelial cells, lymphocytes and epithelial cells in the same variety in compared with the control group, the positive signal of PRRSV infection of pig lungs obviously in the distribution, and in the infection group, the distribution amount of Tongcheng pigs in the lung of PRRSV less than largewhite. (2) through the city and large white pigs The infection of 12 PAM samples before and after PRRSV for high-throughput sequencing of small RNA group, each sample obtained at least 0.5G data obtained at least 10327597 original sequence alignment, mature mi RNA 391 known pig, 67 of them in different combinations. Differentially expressed in 12 pigs as PAM sequencing samples, construct 12 C DNA library after total RNA extraction, for high-throughput sequencing of small RNA group on Illumina Hi Seq2000 platform, the original data of each sample obtained at the volume of more than 0.5G, at least 10327597 original series, more than the number of pure reads sequences obtained after filtration were accounted for 95% of the number of the original sequence of total reads, about 20bp the base error rate is less than 0.02%. The use of MI RDeep2 and Gene Bank in the sequencing sequence of the pig genome sequence and MI RBase mi RNA in the mature sequence alignment, found that 391 reported swine are in 12 samples 鐔焟i RNA.涓や袱緇勫悎T媯，

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