【作者】 殷华平；

【导师】 郭万柱；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 猪细小病毒（porcine parvovirus，PPV）为引起母猪繁殖障碍的主要病原之一，猪细小病毒感染的主要特征是孕猪在怀孕前容易受到感染，可引起胚胎或胎儿的感染和死亡，导致母猪发生流产、死胎、胎儿木乃伊化及新生仔猪的死亡。PPV感染普遍存在于世界各地的猪群中，目前我国PPV的感染呈上升趋势。诊断、预防和控制PPV的流行对于我国的养猪业的发展具有重要的意义。 本文从流产的死胎及木乃伊胎中分离出了一株病毒PPV-SC1，该株病毒能在猪肾原代细胞、PK-15细胞、ST细胞和IBRS-2细胞上增殖并引起细胞病变，出现细胞的聚集、融合现象；分离毒能凝集0.5％的豚鼠红细胞，血凝效价可达1：2¹¹，血凝抑制效价可达1：2¹⁰；荧光抗体染色在细胞核内和细胞质中均可见到特异性荧光，呈苹果绿色；电镜正染观察病毒粒子为无囊膜、球形、大小约23nm的病毒粒子。 根据GenBank中PPV NADL-2株病毒基因组序列用Oligo6.0设计了一对引物，以抽提的PPV-SC1 RF-DNA为模板，通过PCR扩增出长约2.2 kb的DNA片段，将其插入克隆载体质粒pMD 18-T的EcoRV酶切位点处，构建重组质粒pTNS1，对重组质粒pTNS1进行序列测定，测序结果显示PPV-SC1 NS1基因全长1989bp，包含完整的PPV-SC1 NS1基因的开放阅读框架，共编码662个氨基酸。 将PPV-SC1 NS1序列与其他PPV NS1基因进行多序列比对，结果显示，PPV-SC1 NS1与其他的PPV NS1的同源性较高，仅存在个别的差异，分别是第39位A→G，第153位T→C，第175位A→G，第1117位A→C，第1535位A→C；同源搜索比较表明，PPV-SC1与PPV NS1同源性可达98％、99％，与其他的细小病毒NS1基因也存在很大的保守性；密码子偏向性分析结果表明PPV-SC1 NS1基因在同一氨基酸的不同密码子的选择上存在一定的偏向性；PPV-SC1 NS1蛋白总体上说具有亲水性不存在明显的疏水性区段，用swiss TMPRED软件预测PPV-SC1 NS1的跨膜区，返回的结果并没有得到有显著意义的跨膜区的存在；根据基于motif数据库的结构域预测，PPV-SC1 NS1的第393-415位氨基酸残基存在潜在的ATP／GTP结合位点，该蛋白还存在16个蛋白激酶C磷酸化位点，21个酪蛋白激酶2磷酸化位点，3个cAMP-／cGMP依赖蛋白激酶磷酸化位点，PPV-SC1 NS1蛋白与POX_D5（痘病毒D5蛋白）具有一致的保守结构域，推测NS1可能与POX_D5有类似的功能。更多还原

【Abstract】 Porcine parvovirus (PPV) is a major cause of reproductive failure in swine, manifested as embryonic resorption, fetal mummification, abortion and stillbirths .The virus is ubiquitous among swine throughout the world and is enzootic in most herds that have been tested. PPV infection has been increasing in China now. It is important for Chinese stockbreeding to identify ,prevent and control the PPV infection.In this research, we isolate one porcine parvovirus(ppv) strain successfully from the embryonic resorption ,fetal mummification .abortion and stillbirth which can reproduce on swine kidney primary cell,PK-15,ST and IBRS-2 cell lines ,and produce prominence CPE: the infected cells appear gathering and amalgamation.This isolate strain can agglutinate the RBC of guinea pig ,and can be inhibited by the positive ppv serum. Fluorescence assay is used to detect the antigens of porcine parvovirus(PPV),with bright green fluorescence in the infected cells .Under electron microscope ,ppv is 23nm in diameter.without envelope. According to these asseys, we isolated a porcine parvovirus (ppv) strain, named ppv-scl.One pair primers are designed according to the referried genome of ppv NADL-2 strain by Oligo6.0.After amplying a 2.2kb fragment form the PPV-SC1 RF-DNA,we clone the fragment into pMD 18-T,named pTNSl.The whole sequence which is 1989 bp long was determined by sequencing, including the complete ORF of PPV-SC1 NS1 which encoding 662 amino acids.Alignment of pairs of sequence indicates that there are 98% and 99% similary with other porcine parvovirus strains Kresse and NADL-2, respectively. Multiple sequence alignment discloses that there are a few difference between ppv-scl nsl gene and other ppv nsl gene: A-G at 39nt,T-C at 153nt,A-G at 175nt, A-C at 1117nt, A-C at 1535nt .Alternative codon in ppv-scl nsl have distinctly different frequentfy by codonbias analysis at EMBOSS(http://genopole.toulouse.inra.fr/bioinfo/emboss). Thereis not distinct hydrophobicity and transmenbrane helices in ppv-scl nsl protein. Struction domain anslysis of PPV-SC1 NS1 protein indicate that there are a ATP/GTP-binding site motif A (P-loop) at 398-405,16 Protein kinase C phosphorylation site,21 Casein kinase II phosphorylation site,and 3 cAMP/cGMP-dependent protein kinase phosphorylation site.At the same time ,there is a same motif between ppv-scl nsl and Poxvirus D5 protein-like which may share in the same fuction which is necessary during virion duplication.更多还原