PRRSV的分离鉴定及其对Marc-145细胞型朊蛋白表达影响的研究

发布时间：2018-04-21 04:12

本文选题：猪繁殖与呼吸综合征 + 分离鉴定　；参考：《中国农业科学院》2015年硕士论文

【摘要】：猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种以妊娠母猪严重繁殖障碍以及仔猪呼吸道症状和高死亡率为特征的传染病。PRRS自1987年首次在美国报道后,现已几乎遍及世界各地,并成为危害养猪业最严重的传染病之一。与其它囊膜RNA病毒一样,PRRSV具有高度的变异性,不同毒株序列之间存在不同程度的差异,而欧洲型和美洲型毒株之间的差异则更大。本研究从2008年青海某猪场出现高热和呼吸困难等临床症状的发病猪场采集肺脏和血液等样品,在Marc-145细胞上进行病毒培养。将出现特征病变的细胞收集后继续培养并收集病毒。利用RT-PCR、免疫荧光、电镜等方法对分离的病毒进行鉴定,检测结果表明分离的病毒为美洲型PRRSV,将其命名为QH08株。针对PRRSV QH08株设计7对特异性引物扩增全长基因组,对扩增出的片段进行拼接,结果显示PRRSV QH08株基因组全长为15320bp。通过序列比对发现,QH08 PRRSV与高致病性PRRSV(HP-PRRSV)代表株JXA1的全长基因组核苷酸相似性最高,其同源性为98%,并且在NSP区域有两处不连续的90个核苷酸的缺失,所以分离的QH08株属于美洲型HP-PRRSV。为研究QH08毒株对Marc-145细胞的致病性以及毒力强弱等特性,本实验以QH08毒株、海利和诺倍威CH-1R经典毒株、GW-HR毒株在Marc-145细胞上进行传代培养,并对其传代毒株的TCID50高低以及CPE出现的时间进行比较分析。研究结果表明:(1)不同PRRSV株传代毒10个代次的TCID50总趋势为渐变上调的趋势,即随着PRRSV代次的增加,病毒在Marc-145细胞中的增殖速率加快,对细胞的适应性和毒力都增强,与其它三个毒株相比,青海株是毒力最强的。(2)四株PRRSV 10个代次间CPE出现的时间总体呈逐渐缩短的趋势,证明了随着PRRSV毒株代次的增加,病毒在Marc-145细胞中的复制周期缩短,表明不同毒株PRRSV的F10比F1在Marc-145细胞中具备更好的复制能力和致病性。本研究同时进行了PRRSV感染Marc-145细胞后对细胞型朊蛋白(PrPC)表达影响的初步研究。PrPC是一种典型的、具有GPI锚定点的脂筏膜蛋白,在哺乳动物体内各系统中广泛表达。研究表明PrPC可以作为布氏杆菌的一种侵入受体。本研究以QH08毒株和Marc-145细胞为研究对象,通过流式细胞术和qPCR实验(从蛋白水平和基因水平)研究了PRRSV感染Marc-145细胞前后,细胞表面PrPC表达模式的改变。研究发现PRRSV感染Marc-145细胞后,PrPC的表达模式发生显著改变。在PRRSV感染Marc-145细胞后,PrPC的表达在12h时发生显著改变,几乎升高了三倍,然后又迅速恢复到初始水平。而已知的PRRSV的受体-CD163,其表达水平在48h时达到最高值,然后缓慢降低。本研究第一次将PRRSV和PrPC联系在一起,研究两者的关系;首次报道了PrPC可能在病毒侵染宿主细胞中起作用,为PrPC在病毒感染中的生理机能提供了新的视角,也为PRRSV的防治提供新的理论依据。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

【共引文献】