【作者】 布日额；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2005， 博士

【摘要】 小鹅瘟(Goose plague, GP)是由鹅细小病毒(Goose parvovirus,GPV)所引起的主要侵害4～20 日龄雏鹅的急性或亚急性败血性传染病,其造成的死亡率高达90-100%,是危害养鹅业最严重传染病之一。自1956 年中国学者方定一在我国扬州地区发现并进行首次报道后,世界许多养鹅国家和地区均有陆续报道。GPV 属于细小病毒科(Parvoviridae)、细小病毒属(Parvovirus)的成员。病毒整个基因组为5.1kb,含有两个阅读框,左侧阅读框编码NS1、NS2 非结构蛋白,右侧阅读框编码VP1、VP2 及VP3 结构蛋白,其中VP3 结构蛋白能够诱导机体产生中和抗体,因此VP3 基因片段是研究防治小鹅瘟基因工程苗和检测抗原的首选基因。在防治小鹅瘟实践中,GPV 全毒疫苗曾发挥了重要作用,但全毒苗的应用的确存在散毒、潜伏感染,以及对GPV 自然感染与GPV 全毒苗免疫抗体无法进行鉴别等不足,致使该病长期以来无法得到有效检测,直接影响到对小鹅瘟的有效控制。国内外学者先后建立了琼脂扩散试验、中和试验、免疫荧光抗体试验等血清学诊断方法,在一定程度上为防治小鹅瘟发挥了重要作用,但同时也存在灵敏度差、操作烦琐、检测所需时间长等不足,已不适应更有效地控制该病的需要。国外学者建立了根据GPV 核酸PCR 产物酶消化片段的大小来区别小鹅瘟与番鸭细小病毒感染的方法,但未见进一步深入应用的报道。本论文以建立完善、有效的分子生物学和免疫学检测方法,更加有效地控制小鹅瘟为目的,应用扩增GPV 部分核酸片段的特异性引物及其克隆产物分别研究和建立了对GPV DNA进行定性和半定量检测的PCR 及DIG 核酸标记探针方法。利用部分核酸片段的原核表达产物研究建立了定量及定性检测GPV 感染抗体及鉴别VP3 亚单位抗体的间接ELISA 及斑点ELISA 方法。本论文的研究内容和结果如下: 1. 克隆了GPV H1 株的VP1-VP3 核苷酸非重叠序列片段。测序结果为,克隆产物由534bp个核苷酸组成,编码178 个氨基酸残基。扩增产物测序结果与Gen Bank 公布的GPV B 参考毒株同区段序列同源性达100%。2. 利用扩增VP1-VP3 及NS1 核酸片段的两对引物建立了检测GPV 核酸的PCR 方法。两对引物只扩增GPV 模板DNA,对GPMV、AIV 等对照病毒核酸模板的PCR 呈阴性。扩增VP1-VP3 及NS1 核酸片段引物分别能从系列稀释至4.4fg、44pg 或0.01LD50、10LD50的GPV 感染鹅脏器DNA 中扩增出GPV DNA。对7 组感染鹅脏器检测结果表明,两对引物均能快速、准确地扩增出病鹅脏器中的GPV DNA,阳性检出率达100%。2001-2004 年间对多起送检病鹅的PCR 检测均获得一致的结果。但由于扩增NS1 片段引物的检出灵敏度比扩增VP1-VP3 核酸片段引物低1000 倍,因此选择扩增VP1-VP3 核酸片段的一对引物作为建立GPV PCR 检测方法的特异性引物。由于PCR 具有强大的扩增效应,能够从病鹅脏器提取DNA中直接扩增GPV DNA,从而为准确地检测GPV 提供了一项快捷、有效的方法。3. 对VP1-VP3 及NS1 核苷酸片段的扩增产物利用Digoxigenin 进行标记,建立了检测GPV 核酸的DIG 标记探针方法。两个标记探针均能与各自相应核酸片段PCR 产物、重组质粒及不同毒株的GPV 核酸发生特异性杂交,而与GPMV、AIV 核酸杂交呈阴性。VP1-VP3更多还原

【Abstract】 Goose plague (GP) was an acute and sub-acute septicemic infection disease in 4-20 day old gosling by caused goose parvovirus (GPV). Infected goslings have 90-100% of mortality rate. It was one of the important infectious diseases for gosling. This disease was described first in China by Fang Ding-Yig at Yang Zhou district in 1956, followed by reports in various countries in the world. GPV has been classified as a member of the parvovirus genus of the parvoviride. GPV contains a single DNA molecule of about 5.1kilobases in length. The genome of GPV contains two major open reading frames (ORF),left one encoding for the non-structural proteins NS1 and NS2 and the right one encoding for the VP1, VP2 and VP3 structural proteins. VP3 was the major structural protein, as well as it was a major protective antigen and enable to induce the body to produce neutralizing antibodies. So the genes of VP3 were the important candidate genes for study on genetic engineering vaccine and detecting antigen. GPV inactivated and attenuated whole virion vaccines were be used to prevent GPVI, but these vaccines have some side-effects to spread virus and to cause sub-clinical infection. So this disease has not been effectively prevented. It was the main cause that lacking of fast and effective diagnostic and identifing methods of GPVI has not been effectively controlled. The routine diagnostic methods for GPVI were the Agargel precipitin assays (AP),Serum-neutralization test (NT),Immunofluorescent antibody assay (IFA) have been established. These methods have played important role in control GP, but which have lower sensitivity and use longer time. These methods have not suitable for the needs of efficiently control GP. A PCR method was established to identify GPV and GPMV by the digested fragments, but have not found the following reports about using this method. This study aimed at established effective and fast molecular biological and immunological identifying methods of efficiently control GPVI. PCR and DIG-labeled nucleic acid probes detecting methods for quantification and half-quantification were developed by two pairs of primers of VP1-VP3, NS1 nucleotide and by it’s amplified products. To detect method of Indirect-ELISA and Dot-ELISA were developed by the expressed products of some nucleic acid fragment for quantify and determine GPV antibody. The results of this study showed that: 1. Cloning VP1-VP3 non-repeated nucleic acid sequences and obtaining products of PCR about 0.6kb in length. The sequencing result showed that the product of PCR contains 534 bp, which encodes 178 amino acid residues. The nucleotide and amino acid sequences have 100% homology with same region nucleic acid of GPV B reference strain. 2. The PCR detection methods were developed by two pairs of primers to amplify VP1-VP3 and NS1 nucleic acid. The specificity assay showed that two pairs of primers disable to amplify the control nucleotide of GPMV and AIV. Sensitivity assay showed that the pair of primers of VP1-VP3 nucleotide can detect 4.4fg (0.01LD50 GPV)of GPV nucleic acid, and the other pair can detect 44pg((0.01LD50 GPV) of GPV nucleic acid. The result of detecting 7 groups organs from GPV infected goose showed that two pairs of primers can fast and efficiently detect GPV DNA, and the positive rate was 100%. But the detecting sensitivity of NS1 nucleic acid primers was less than VP1-VP3 primers of 1000 times, so that the pairs of primers to amplify VP1-VP3 nucleotide fragment were selected to develop PCR detecting method. PCR have a strongly amplifying function, which could directly to detect GPV DNA from the extracted DNA of GPV infected gooses, so PCR was an effective and fast method to detect GPV. 3. The amplified product of VP1-VP3 nucleotide and NS1 nucleotide were labeled with Digoxigenin and developed the DIG-labeled nucleic acid probes to detect GPV DNA. The specificity assay showed that two labeled probes all enable to hybridize with target nucleotide products of PCR, DNA recombinant plasmid and nucleic acid from different GPV strain, and disable to hybridize with the control nucleic acid of GPMV and AIV. The sensitivity assay showed that NS1 and VP1-VP3 DIG labeled nucleic acid probes were able to detect 6.6pg and 9.9pg of GPV DNA respectively. The result of using two labeled probes to detect 2 groups organs from GPV infected goose showed that positive rate was 100%, which indicate that DIG-labeled probe was a fast detecting, suitable detecting large number samples one time, un-use specific equipment and suitable popularized method to diagnose GP. 4. The sensitivity of DIG-labeled probe method was less than PCR method. The sensitivity (4.4fg) of PCR method by VP1-VP3 primers was higher of 2250 times than the sensitivity (9.9pg) of DIG-labeled probe method. The sensitivity (44pg) of PCR method by NS1 primers was lowerr of 6.7 times than the sensitivity (6.6pg) of DIG-labeled probe method. 5. pGEX-(VP1-VP3) recombinant expressive plasmid was constructed and over expressed in GST fusion proteins. The suitable expression and purification conditions were confirmed. The provided recombinant plasmid pGEX-VP1, pGEX-VP2, pGEX-VP3, pGEX-VS1 and pGEX-NS2 by this laboratory were also expressed and purified successfully by the Prepared Glutathion Sepharos 4B after renaturing the expressed products. According to the result of Western blot and Dot-ELISA, selecting VP3 protein as the antigen to develop the Indirect-ELISA and Dot-ELISA method for quantifying and determining GPV antibody, selecting VP1-VP3 protein as the antigen to develop the Indirect-ELISA and Dot-ELISA method for identifying GPV antibody and VP3 sub-unit antibody. The result was satisfactory after detecting antibody titer of 90 serum samples from immunized with whole GPV vaccine. The result was relative to the neutralizing test antibody titer. All the above, these detecting method have been developed, which indicated that the high technological products of the cloned nucleic acid and expressed products of GPV structural proteins and non-structural proteins gene were have been applied to practical and were have beenused to produce economic returns. The PCR and labeled probe detecting methods were suitable for determining the GPV nucleic acid before antibody produce, and the ELISA methods were suitable for detecting infected antibody and determining immunized antibody. These methods have important practical significance and use value for fast diagnosis, control epidemic situation, epidemiological investigation, detecting immunizing antibody titer, identifying natural infection, and dispelled the trouble about identifying infection antibody when using VP3 gene engineering vaccine, which lay the academic and technical foundation for further development and assemble detecting reagent boxes.更多还原