【作者】 赵俊龙；

【导师】 陈焕春； 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2001， 博士

【摘要】 猪细小病毒病是由猪细小病毒引起的以初产母猪发生流产、不孕、产死胎、畸形胎、木乃伊胎及弱仔等为特征的猪的主要繁殖障碍性疾病之一。该病广泛存在于世界各地并在大多数猪场流行，严重地影响着养猪业的发展，因此一直是猪病研究的热点之一。本研究首先建立了用于猪细小病毒感染检测的PCR方法，在此基础上利用PCR技术扩增了猪细小病毒的近全基因组序列并与相关的毒株进行了比较和分析，分别克隆了结构蛋白基因VP₁和VP₂，进行了原核表达、真核表达以及基因免疫的研究。 根据已报道的猪细小病毒基因组序列，设计并合成了一对寡核苷酸引物，通过对影响PCR扩增因素的筛选成功地从猪细小病毒感染的组织和细胞中扩增出445bp片段，回收该片段用EcoRI酶切得到了预期的结果，证实了该扩增片段的特异性。敏感性试验表明该方法可以检测出10^-4个TCID₅₀的病毒含量。利用该方法对临床上24份流产病料的检测，查出阳性16份，而同时利用HA检测阳性只有10份。这些结果说明本研究建立的PCR诊断方法具有灵敏度高、特异性强的特点。 以已经建立的PPV和PRV的单项PCR诊断方法为基础，通过对扩增条件的筛选，成功地建立了PPV和PRV的二联PCR诊断方法，并应用于临床，利用一次PCR反应，即可同时扩增PPV的445bp和PRV 217bp的特异性片段。该方法的建立对临床上进行这二种疾病的鉴别诊断和混合感染的检测都具有重要意义。 以感染细胞中提取的RFDNA为模板，利用2次PCR反应扩增了包含猪细小病毒的近全基因组的DNA片段并进行了克隆和测序。将该序列在GenBank网站上利用DNA Blast软件进行比较发现该株病毒与GenBank收录的PPV基因组的核苷酸和氨基酸同源性均达到98％以上，证明了PPV基因组序列是十分保守的。从比较中可以看出，中国株PPV基因组的结构和已报道的PPV基因组的结构一致，即整个基因组由2个主要的开放阅读框架组成，5′端编码非结构蛋白NS₁、NS₂、NS₃，3′端编码结构蛋白VP₁、VP₂，整个编码区基因互相重叠，NS₂重叠在NS₁内，NS₃重叠在NS₁和VP₁内，VP₂重叠在VP₁内。 结构蛋白VP₁和VP₂是猪细小病毒的主要免疫原性蛋白。本研究利用PCR技术从猪细小病毒的RF DNA模板中扩增了含有VP₁和VP₂全基因的2.5kb和2.0kb的基因片段，将PCR产物克隆至pMD18-T载体，利用双脱氧末端终止法测定了VP₁和VP₂全基因的核苷酸序列。将所得的核苷酸序列和推导的氨基酸序列分别与GenBank中的相应序列进行同源性比较，同源性均在99％以上，从而说明这两种蛋白的碱基序列和氨基酸序列均具有高度的保守性。华中农业大学博士学位论文 将vPZ全基因分别克隆入原核表达载体pETzs（b）、pET17（b）和pET28（b）构建成表达质粒pET15bVpZ、pET17bVp:和pET28bvPZ;将含有VP:基因起始位点至&o班切点间的o.skb片段克隆入pET17（b）中构建成表达质粒pET17bvPZfo利用上述四种质粒转化大肠杆菌BLZ:，IPTG诱导后SDS一PAGE检测表达情况，结果发现pET17bvPZf质粒在45kD处有一特异性表达带，而其它几种质粒均未看到特异性表达带，推测VPZ基因3’端的某些结构可能对VPZ全基因的表达有一定影响。这一结果对研究VPZ基因的结构与功能具有重要意义。 利用真核表达载体peDNA3 .1（+）和pChico分别构建了含有vPI基因的pChieovPI和含有vPZ基因的pChieovPZ和PcDNAVPZ三种真核表达质粒。将上述三种真核表达质粒分别转染IBRS一2细胞，利用间接ELISA检测表达情况，结果表明上述三种质粒均能在IBRS一2细胞表达，表达产物位于细胞中。在此基础上，利用这三种质粒分别以肌肉注射的方式间隔2周2次免疫小鼠，结果发现所有表达质粒均能诱导产生明显的细胞免疫和体液免疫，其中pCIneovP、质粒诱导的体液免疫最强，和PPv灭活苗免疫组相当，pCIneovPZ诱导的细胞免疫反应强于PPv灭活苗组，PChieovP:和pChicoVP:联合免疫并没有加强作用。这一结果预示着PPv基因免疫的良好前景。有关基因免疫诱导的免疫保护作用还有待进一步研究。 本研究中PPV PCR和二联PCR检测方法的建立解决了PPV持续感染和传代细胞系PPV污染不易检测的难题，对猪细小病毒病的临床诊断、控制以及生物制品的生产等均具有重要意义。中国株细小病毒全基因组序列的首次测定则为进一步研究细小病毒的分子生物学打下了坚实的基础。结构蛋白基因的克隆、表达及基因免疫的研究则对猪细小病毒病的免疫诊断、分子流行病学、免疫预防以及抗病育种研究具有重大价值。更多还原

【Abstract】 Porcine parvovirus (PPV) causes 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. In this research, the PCR method for the detection of PPV was developed. The genome DNA was sequenced and comparison with those of NADL-2 and Kresse strains were carried out. Then the structural protein gene VP1 and VP2 were cloned, sequenced, expressed and the possibility of DNA vaccine were studied too.A pair of primers was derived from the DNA sequences that have been published. The PCR method was developed for the detection of PPV DNA. The results revealed that the expected 445 bp fragment could be amplified from many kinds of tissues of infected foetus and passaged cells. The amplified fragment was shown to be specific for PPV DNA after digested by EcoRI. This method could detect at least the virus of 10-4 TCID50. 16 positive of 24 samples from clinical sick swine were detected by PCR and only 10 positive were detected by HA test. Theses results showed that the PCR method had the specificity and high sensitivity.On the basis of single PCR of PPV and PRV, multiple PCR was established. The results showed that two specific fragments of 445bp for PPV and 217bp for PRV could be amplified in one PCR. This method could be differentiate PRV infection, PPV infection and mixed infection from reproductive failure.The geneome DNA of PPV has been amplified by two PCR amplifications and the sequence of amplified fragment has been determined. The nucleotide sequence organization of the viral genome was found to be similar to that of the other PPV strains and the homology was high over 98%. The sequence analysis showed that there were two open reading frames (ORFs) in PPV genome. The left ORF had the coding domains for all of NS1 NS2 and NS3. The right ORF had the coding domains for both VP1 and VP2.NS3, NS2 gene were overlapped with NSt gene, VP2 gene were overlapped with VP1 gene.The VP1 and VP2 fragments were amplified from PPV RFDNA by PCR method. Then the products were cloned into pMD18-T vector and the sequence were determined. The homology analysis of these genes with others reported in GenBank showed that these gene were high conserved.The completed VP2 gene were cloned into pET15(b), pET17(b) and pET28(b) vectors respectively and 0.8kb fragment of VP2 gene were cloned into pET17(b) vector, the recombinant plasmid pET15bVP2, pET17bVP2, pET28bVP2 and pET17bVP2f were constructed. Then these plasmids were introduced into E. coli BL21. After induction by IPTG, a high expression was found in products of pET17bVP2f, while no expression protein was detected for other plasmids. These results will be useful for further research of VP2 gene.The completed VP1 and VP2 gene were cloned into the the eukaryotic expression vector of pcDNA 3.1(+) and pCIneo, resulting recombinant expression plasmid pcDNA VP2, pCIneo VP2 and pCIneo VPi. Then these plasmids was transfected into IBRS-2 cells and several clone cells were obtained under the selection of G418. Expressed protein was detected by ELISA. On the basis of above work, these plasmids were used as DNA vaccine to immune mouse. The results showed that all these plasmids could induce specific cell-mediated and humoral immunity. The specific cell-mediated immunity induced by pCIneoVP2 was stronger than that induced by inactived PPV vaccine, the humoral immunity induced by plasmid pCIneoVP1 was stronger than that induced by inactived PPV vaccine. The immune responses induced by co-immunization with pCIneo VP1 and pCIneo VP2 were not stronger than that induced by pCIneo VP2 or pCIneo VP1 alone. This preliminary results showed hope of developing PPV DNA vaccine and worthy to continue further studies.更多还原