论文标题:欧洲型PRRSV的分离与鉴定及其ORF5基因变异分析
Isolation and Identification of Europe Type PRRSV and Variation Analysis of Its ORF5 Genes
论文作者
论文导师 杨增岐；宋长绪,论文学位 硕士,论文专业 预防兽医学
论文单位 西北农林科技大学,点击次数 106,论文页数 77页File Size17513K
2006-06-01论文网 http://www.lw23.com/lunwen_64392167/
PRRSV; European type; Identification; ORF5 gene; Variation analysis
猪生殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome,PRRS)是20世纪80年代末发现的一种高度传染性疾病,引起妊娠母猪的流产、死胎、木乃伊胎等繁殖障碍症状,以及各种年龄猪(特别是仔猪)的呼吸道症状,其病原为猪生殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV),属于新成立的尼多病毒目(Nidovirales )、动脉炎病毒科(Arteriviridae )、动脉炎病毒属(Arterivirus)。PRRSV基因组长约15 kb,包括8个开放阅读框架(ORFs),ORFla和ORFlb编码病毒复制酶,ORF2～5分别编码病毒的糖蛋白GP2～GP5,ORF6编码膜蛋白(M蛋白),ORF7编码保守性的核衣壳蛋白(N蛋白)。 PRRSV的GP5是该病毒最重要的结构蛋白之一,具有良好的抗原性,另外,ORF5是PRRSV基因变异最明显的阅读框。根据抗原性不同将各地PRRSV分离株分为欧洲型(代表毒株为LV)和北美洲型(代表毒株为VR-2332),前者主要流行于欧洲地区,后者主要流行于美洲和亚太地区。然而加拿大和美国研究人员于2000年分别在各自国家分离到类似LV的PRRSV。目前,已发表的中国大陆分离的PRRSV毒株均为美洲型。那么,中国大陆有没有欧洲型PRRSV的感染呢?目前还未见系统的调查报告。本研究以实验室现有的技术和材料为基础,首先用肺泡巨嗜细胞和MARC-145细胞对疑似患有PRRS的猪病料作病毒分离,并用特异性单克隆抗体鉴定分型,以确定中国是否存在欧洲型PRRSV。 首先用肺泡巨嗜细胞和MARC-145细胞从临床诊断为PRRSV感染猪的血液,淋巴结等病料中分离到17株病毒,并用既能识别PRRSV欧洲株又能识别PRRSV美洲株单克隆抗体M1对分离的病毒进行鉴定,再用能特异识别PRRSV美洲株NSP2蛋白的单克隆抗体M2和能特异识别PRRSV欧洲株NSP2蛋白单克隆抗体M3对用M1鉴定为阳性的病毒进行分型鉴定,结合荧光免疫技术证实共分离到17株PRRSV,其中有9株为欧洲型PRRSV,占分离毒株的52.9%。然后根据GeneBank中猪生殖与呼吸综合征病毒欧洲毒株(LV株)基因序列分别设计合成了针对ORF5基因(含部分ORF4和ORF6基因)的引物,利用RT-PCR方法从9株欧洲型分离毒株细胞培养物中扩增出大小约830 bp片段,克隆入pMD18- T载体后,送往宝生物工程技术有限公司测序。最后应用DNAStar软件对所测的基因序列与国内外已发表毒株的序列进行同源性比较,构建基因进化树,并对其推导氨基酸序列及预测蛋白的一些特性与国内外已发表毒株进行比较。分析结果表明9株分离毒株与LV等欧洲型分离毒株亲缘关系较近,推导氨基酸序列同源性在92%以上,尤其与几株西班牙毒株亲缘关系最近,推导氨基酸序列同源性在95%以上,并且抗原性非常
Porcine reproductive and respiratory syndrome virus(PRRSV) has been one of the most important pathogens of swine science. Its emergence in the late 1980s.The disease has many clinical manifestations ,but the two most prevalent are severe reproductive failure in sows and gilts(characterized by late-term abortions, an increased number of stillborn, mummified and weak-born pigs),and respiratory problems in pigs of all ages. PRRSV belongs to the recently recognised Arterivirdae family within the genus Arterivirus, order Nidovirales. The virus is comprised of a 15 kilobase (kb) single-stranded positive sense RNA genome that is encapsulated within an enveloped, icosahedral-shaped nucleocapsid. The replicase gene covers the 5’-terminal four-fifths of the genome and is composed of two overlapping open reading frames, ORF1a and ORF1b,which are expressed from the genomic RNA. The structural genes encoded by ORF2 to 7. GP5 proteins are the most important structural protein, which is associated with immunity. According to the difference of the antigen, the isolations had been divided into the North American and European strains, the former is mostly epidemic in America and Asia, the latter is mostly epidemic in Europe, but Canadian and American researchers had isolated European strains in their country. At the present time, PRRSV had been isolated in China are all North American type. So,is there any European type PRRSV in China? There is not systemic investigation report about it at present. This research base on the technology and material of our lab, using PAMs and MARC-145 cells to isolate virus from tissue of pigs that diagnosis is the PRRSV infection, and using monoclonal antibody to identify virus in order to confirm that is there any European type PRRSV existing in China . First of all, using PAMs and MARC-145 cell, 17 virus were isolated from bloods, lymph node and lungs of pigs that diagnosis is the PRRSV infection, and using monoclonal antibody M1 that both can distinguish European type and American type PRRSV to identify virus that have been isolated, and then using monoclonal antibody M2 that can specially distinguish PRRSV Americas Nsp2 protein and monoclonal antibody M3 that specially distinguish PRRSV European Nsp2 protein to affirm the type of them. Combining fluorescence immunity
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