【作者】 魏战勇；

【导师】 黄克和； 崔保安；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2004， 博士

【摘要】 猪细小病毒（Porcine Parvovirus，PPV）自二十世纪六十年代被发现以来，许多国家先后分离到该病毒和检查到其抗体，抗体检出率为50％～80％，其中我国高达80％。该病毒感染能引起以母猪繁殖障碍为主要特征的病毒性传染病，孕前期受到感染时，经胎盘使胚胎或胎儿受到侵袭，引起母猪流产、不孕、胚胎死亡、胎儿畸形及木乃伊化等，同时还可引起仔猪的皮炎和腹泻，给养猪业造成巨大的经济损失。本文主要是探讨PPV地方毒株和标准毒株在体外增殖的基本特性与规律，NO和硒对PPV体外增殖的影响，不同毒株和不同代次的分子变异规律，研制PPV的核酸疫苗，以期为猪细小病毒病发病机理的阐明和临床上该病的防治提供实验依据。研究內容如下： 1．应用免疫荧光法较为系统地研究了猪细小病毒南京株1（NJ-1）、南京株2（NJ-2）和7909株在PK-15细胞上增殖的基本特性与规律。结果显示，三个毒株的增殖规律基本相似，均在感染后12h开始产生子代病毒粒子，随后逐渐增多，48h子代病毒遍布所有细胞，随后开始衰减，在84h时病毒粒子引起细胞大面积崩解而释放到培养液中；通过一步法生长曲线可知：在病毒感染后6Dh时病毒的TCID₅₀达到最高，随后逐渐下降；培养液中病毒粒子的半寿期为7h。 2．分别研究了来源于不同前体物的NO和来自不同硒源的硒对PPV在PK-15细胞上增殖的抑制作用。结果首次证明，NO前体物S-硝基-N-乙酰青霉胺（SNAP）、L-精氨酸（L-Arg）均能有效地诱导PK-15细胞产生NO，进而显著地抑制PPV在PK-15细胞上的复制，其效果与前体物的浓度呈正相关，其中在100μmol／L和200μmol／L浓度时SNAP产生NO的能力与抑制病毒复制的作用要强于L-Arg。在病毒感染前6h和3h添加SNAP或L-Arg对病毒复制的抑制作用比在病毒感染后3h和6h添加的作用强，提示NO的抗病毒作用主要发生在病毒感染的初始阶段。此外，添加具有抑制L-Arg产生NO作用的N-硝基-L-精氨酸（L-NNA）能抵消L-Arg体外抗病毒的作用。对硒的研究首次证明：三种不同硒源对PPV在PK-15中的复制呈现不同程度的抑制作用，其强度依次为硒蛋氨酸、亚硒酸钠、海藻硒多糖；而且随着浓度的增加，其抑制作用逐漸增强，呈剂量依赖性关系。自由基清除基还原型谷胱甘肽和甘露醇均有增强硒的抗病毒作用的效果。 3．对PPV不同毒株和同一毒株的不同代次的VP2基因序列差异性进行了分析。参考NADL-2株序列，设计出一对引物，应用PCR分别扩增了NJ-1株、NJ-2株、7909博士学位论文:猪细小病毒体外增殖的抑制因素、VPZ基因变异及核酸疫苗的研究株和vaeeine株的vPZ基因，测序得到NJ一l、NJ一2和7909株均为一438bp，共编码476aa;而vaeeine株为657bp，缺失7slbp，共编码Z19Aa:与Genbank中NAnL一2株，Kresse株、China株及VR一1株相应的核普酸及氨基酸进行同源性比较，发现其核普酸和氨基酸的同源性分别在97.7%-99.9%、97.2%一99.2%之间，具有高度同源性;通过绘制系统进化树可知，来源于国内的ehina株、NJ一1株、NJ一2株、7909株及Vaeeine株亲缘性最为相近，与其它株亲缘性较远.试验还对NJ一1株的第1、7、14、21、28及35代的vPZ基因进行了扩增和测序，均为1438bP，并推导出相应的氨基酸序列，共编码476 Aa;分析发现，核普酸及氨基酸序列同源性分别为997%一100%、99.2%一100%，说明不同代次之间具有高度的同源性，该病毒遗传稳定性强。4.成功地构建了在体外能能够表达VPZ蛋白、在体内能够有效诱导机体产生体液免疫和细胞免疫的pCIneo一VpZ、pCIneo一ILZ一VpZ重组质粒.利用设计出的引物扩增ppVvPZ主要杭原基因，得到882bP的基因片段，并推导出194aa氨基酸序列，应用DNAstar软件对氛基酸的杭原表位进行了预测，共有九个B细胞杭原表位。将该VPZ基因和IL一2基因克隆到真核表达载体pclne。，分别构建了pclne。一ILZ一vPZ和pCIne。一VPZ两种真核表达重组质粒，分别转染到PK一巧细胞，利用直接荧光杭体技术检测表明，两种质粒均能够表达vPZ蛋白，其中pCIneo一ILZ一VPZ质粒还能检测到具有生物活性的IL一2。以小鼠为动物模型，通过肌内注射pclneo一vPZ、pclneo一ILZ一vPZ进行免疫接种，分别用竞争ELISA检测PPV杭体，MT’T法测定机体的淋巴细胞转化功能及细胞毒活性。结果显示，两种重组质粒1周后均能够诱导机体产生杭体，4周时达到峰值，与活疫苗对照组产生的杭体滴度基本一致;两种重组质粒及活疫苗均能够诱导T淋巴细胞增殖，但pclne。一vPZ不诱导B淋巴细胞增殖;两种重组质粒及活疫苗能够诱导较强的细胞毒活性。关键词:猪细小病毒;增殖;一氧化氮;不同硒源;遗传变异分析;核酸疫苗更多还原

【Abstract】 The disease of porcine parvovirus (PPV) is caused by porcine parvovirus which is a major cause of reproduction failure in swine. Parvovirus infections in pigs may cause fetal death and mummification, still births and other reproductive failures in pregnant sows, and dermatitis and diarrhea in piglets. Since PPV was isolated by Carwright in 1967, the virus and it’s antibody were proved to be positive in many countries, and the antibody positive rate of PPV reaches to 50%~80%. In china, the antibody positive rate of PPV was up to 80%, which causes lots of economical losses in swine production. In present study, the proliferous properties of PPV in porcine kidney (PK-15) cells, inhibitory effects of nitric oxide and different selenium sources on PPV infection in vitro, the pathogenicity of different strain of PPV and their rule of variation were studied, which provided theoretic and technological base of prevention and treatment in the disease. Apart from these, the safety and effective DNA vaccination was developed. The research contents are as follows.1. Some proliferation properties of three strains of PPV in PK-15 cells were studied by using the immunofiuorescence technique. The results suggested that the three strains had the similar characteristics of the multiplication. The location of virus was observed in cytoplasm at 12 h post-inoculation, followed by a highest production at 48 h after inoculation. Then the viral particles inside host cells began to reduce and caused host cells to crack, and the particles released from the cells. Based on the results, the one-step growth curve of cells was drawn, which showed that the viral titers were reached to the highest level at 60 h post-inoculation, subsequently the viral concentration began to decrease.2. Inhibitory effects of NO and different selenium sources on PPV infection on PK-15 cells were studied. The result showed that production of NO was effectively induced when the cultures were treated with S-nitroso-N-acetylpenicllamine (SNAP) or L-Arginine, and the replication of PPV on PK-15 cellswas significantly inhibited by the addition of these materials, which have a dose-dependent manner. The anti-PPV effects of L-Arginine could be blocked by the addition of NG-nitro-L-Arginine. Higher level of inhibition of viral production was also obtained when pretreated with SNAP 6 h and 3 h before infection than added 3 h and 6 h after infection. Inhibitory effects of different selenium sources (Sodium selenite, DL-Selenomethionine and Kappa-selenocarrageenan) on PPV infection in vitro on the PK-15 cells were also studied. In addition, the influences of Glutathione reduced (GSH) and D-Mannitol on antiviral effects of selenium were carried out on PK-15 cells. The results showed that all the three kinds of selenium sources have inhibitory effects on the PPV replication in vitro. Among them, DL-Selenomethionine had the highest inhibitory effects, the lowest was the Kappa-selenocarrageenan, and they also produced a concentration-dependent manner. The GSH and D-Mannitol have the ability to increase the antiviral effects of Selenium. The antiviral effect was enhanced obviously with the addition of GSH and D-Mannitol together.3. In order to investigate the pathogenicity of different strain and rule of variation of PPV, VP2 gene of NJ-1 strain, NJ-2 strain, 7909 strain and Vaccine strain of PPV were amplified by PCR, and cloned into pGEM-T Easy respectively and sequenced. Then the differences of these sequences were compared. Among the four sequences, NJ-1 strain, NJ-2 strain, 7909 strain were all 1438 bp, which all encode 476 Aa. The vaccine strain was 657 bp, encoding 219 Aa, which was 781 bp shorter than other strains. The four sequences with NADL-2 strain, Kresse strain, China strain and VR-1 strain coming from GenBank were compared. The nucleotide sequence homologies were 97.7%-99.9% and the amino acid homologies were 97.7%~99.2%. By analysis of phylogenetic tree, it was found that the sequenced strains have higher homology with China strain, but have lo更多还原