發(fā)布時(shí)間：2018-04-21 04:12

  本文選題：豬繁殖與呼吸綜合征 + 分離鑒定�。� 參考：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：豬繁殖與呼吸綜合征(PRRS)是由豬繁殖與呼吸綜合征病毒(PRRSV)引起的一種以妊娠母豬嚴(yán)重繁殖障礙以及仔豬呼吸道癥狀和高死亡率為特征的傳染病。PRRS自1987年首次在美國報(bào)道后,現(xiàn)已幾乎遍及世界各地,并成為危害養(yǎng)豬業(yè)最嚴(yán)重的傳染病之一。與其它囊膜RNA病毒一樣,PRRSV具有高度的變異性,不同毒株序列之間存在不同程度的差異,而歐洲型和美洲型毒株之間的差異則更大。本研究從2008年青海某豬場(chǎng)出現(xiàn)高熱和呼吸困難等臨床癥狀的發(fā)病豬場(chǎng)采集肺臟和血液等樣品,在Marc-145細(xì)胞上進(jìn)行病毒培養(yǎng)。將出現(xiàn)特征病變的細(xì)胞收集后繼續(xù)培養(yǎng)并收集病毒。利用RT-PCR、免疫熒光、電鏡等方法對(duì)分離的病毒進(jìn)行鑒定,檢測(cè)結(jié)果表明分離的病毒為美洲型PRRSV,將其命名為QH08株。針對(duì)PRRSV QH08株設(shè)計(jì)7對(duì)特異性引物擴(kuò)增全長(zhǎng)基因組,對(duì)擴(kuò)增出的片段進(jìn)行拼接,結(jié)果顯示PRRSV QH08株基因組全長(zhǎng)為15320bp。通過序列比對(duì)發(fā)現(xiàn),QH08 PRRSV與高致病性PRRSV(HP-PRRSV)代表株JXA1的全長(zhǎng)基因組核苷酸相似性最高,其同源性為98%,并且在NSP區(qū)域有兩處不連續(xù)的90個(gè)核苷酸的缺失,所以分離的QH08株屬于美洲型HP-PRRSV。為研究QH08毒株對(duì)Marc-145細(xì)胞的致病性以及毒力強(qiáng)弱等特性,本實(shí)驗(yàn)以QH08毒株、海利和諾倍威CH-1R經(jīng)典毒株、GW-HR毒株在Marc-145細(xì)胞上進(jìn)行傳代培養(yǎng),并對(duì)其傳代毒株的TCID50高低以及CPE出現(xiàn)的時(shí)間進(jìn)行比較分析。研究結(jié)果表明:(1)不同PRRSV株傳代毒10個(gè)代次的TCID50總趨勢(shì)為漸變上調(diào)的趨勢(shì),即隨著PRRSV代次的增加,病毒在Marc-145細(xì)胞中的增殖速率加快,對(duì)細(xì)胞的適應(yīng)性和毒力都增強(qiáng),與其它三個(gè)毒株相比,青海株是毒力最強(qiáng)的。(2)四株P(guān)RRSV 10個(gè)代次間CPE出現(xiàn)的時(shí)間總體呈逐漸縮短的趨勢(shì),證明了隨著PRRSV毒株代次的增加,病毒在Marc-145細(xì)胞中的復(fù)制周期縮短,表明不同毒株P(guān)RRSV的F10比F1在Marc-145細(xì)胞中具備更好的復(fù)制能力和致病性。本研究同時(shí)進(jìn)行了PRRSV感染Marc-145細(xì)胞后對(duì)細(xì)胞型朊蛋白(PrPC)表達(dá)影響的初步研究。PrPC是一種典型的、具有GPI錨定點(diǎn)的脂筏膜蛋白,在哺乳動(dòng)物體內(nèi)各系統(tǒng)中廣泛表達(dá)。研究表明PrPC可以作為布氏桿菌的一種侵入受體。本研究以QH08毒株和Marc-145細(xì)胞為研究對(duì)象,通過流式細(xì)胞術(shù)和qPCR實(shí)驗(yàn)(從蛋白水平和基因水平)研究了PRRSV感染Marc-145細(xì)胞前后,細(xì)胞表面PrPC表達(dá)模式的改變。研究發(fā)現(xiàn)PRRSV感染Marc-145細(xì)胞后,PrPC的表達(dá)模式發(fā)生顯著改變。在PRRSV感染Marc-145細(xì)胞后,PrPC的表達(dá)在12h時(shí)發(fā)生顯著改變,幾乎升高了三倍,然后又迅速恢復(fù)到初始水平。而已知的PRRSV的受體-CD163,其表達(dá)水平在48h時(shí)達(dá)到最高值,然后緩慢降低。本研究第一次將PRRSV和PrPC聯(lián)系在一起,研究?jī)烧叩年P(guān)系;首次報(bào)道了PrPC可能在病毒侵染宿主細(xì)胞中起作用,為PrPC在病毒感染中的生理機(jī)能提供了新的視角,也為PRRSV的防治提供新的理論依據(jù)。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a kind of infectious disease characterized by severe reproductive disorders of gestation sows and respiratory symptoms and high mortality of piglets. Since 1987, the.PRRS has been reported in the United States for the first time in the world, and has become the most serious harm to the pig industry. One of the infectious diseases. Like other membrane RNA viruses, PRRSV has a high variability. There are different degrees of difference between the sequence of different strains, and the difference between the European and American type strains is greater. This study collected the lungs and blood from the swine farm which had high fever and dyspnea in 2008 in a pig farm in Qinghai. The samples were cultured on the Marc-145 cells. The cells of the characteristic lesions were collected to continue to be cultured and collected. RT-PCR, immunofluorescence and electron microscopy were used to identify the isolated viruses. The results showed that the isolated virus was American PRRSV, and its life was named QH08 strain. 7 pairs of specificity against PRRSV QH08 strain were designed. The primers amplified the full-length genome and spliced the amplified fragments. The results showed that the total length of the genome of PRRSV QH08 strain was 15320bp. through sequence alignment, and the highest similarity between QH08 PRRSV and highly pathogenic PRRSV (HP-PRRSV) represented the full-length genome of JXA1, and its homology was 98%, and there were two discontinuities in the NSP region of 90. The deletion of nucleotides, so the isolated QH08 strain belongs to the Americas type HP-PRRSV. to study the pathogenicity and virulence of the QH08 strain to Marc-145 cells. In this experiment, the QH08 strain, the classic strain of Haili and Nobel CH-1R, the GW-HR strain on the Marc-145 cells, and the TCID50 high and low of its passages and the CPE out of the CH-1R strain. The results showed that: (1) the general trend of the 10 generation of PRRSV strains of different generations was the trend of up regulation of TCID50, that is, with the increase of PRRSV generation, the proliferation rate of the virus in Marc-145 cells is quicker, the adaptability and virulence of the cells are enhanced. Compared with the other three strains, the Qinghai strain is the most virulent. Strong. (2) the time of the occurrence of CPE in 10 generations of PRRSV four strains was gradually shortened, which showed that with the increase of PRRSV strain, the replication cycle of the virus in Marc-145 cells shortened, indicating that the F10 of different strains of PRRSV had better replication ability and pathogenicity than F1 in Marc-145 cells. Preliminary study on the effect of infection of Marc-145 cells on the expression of cell type prion protein (PrPC),.PrPC is a typical type of lipid rafting protein with GPI anchorage point, which is widely expressed in various systems in mammals. The study shows that PrPC can be used as a kind of invasive receptor of brucellus. This study is based on QH08 and Marc-145 cells. Like, through flow cytometry and qPCR experiment (from protein level and gene level), the expression pattern of PrPC in the cell surface was changed before and after PRRSV infection of Marc-145 cells. The study found that the expression pattern of PrPC was significantly changed after PRRSV infected Marc-145 cells. After PRRSV infected Marc-145 cells, the expression of PrPC was significantly changed in 12h. It was changed, almost three times higher, and then quickly recovered to the initial level. And the known PRRSV receptor -CD163, whose expression level reached the highest value at 48h, then slowed down slowly. The first time this study was associated with PRRSV and PrPC to study the relationship between the two, the first report that PrPC may play a role in the virus infecting host cells, for the first time, PrPC The physiological function of virus infection provides a new perspective and provides a new theoretical basis for the prevention and treatment of PRRSV.

【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 秦運(yùn)杰;CD163分子在PRRSV-ADE中的作用[D];河南農(nóng)業(yè)大學(xué);2014年

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本文編號(hào)：1780869