【作者】 马波；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2002， 硕士

【摘要】 GPV（Goose Parvovirus）依据其形态、生化及培养特性被划归为细小病毒科细小病毒属成员，引起雏鹅和雏番鸭高度致死性疾病，主要以消化道，尤其是小肠部位的典型病变为特征。许多国家都爆发过该病，所有分离的GPV抗原性相似。目前主要依据临床症状结合血清学、病毒学方法诊断该病，这些方法大多费时、费力，容易延误该病的治疗，需建立一种快速、简便、特异的诊断方法。该病的防制依赖疫苗和抗血清及卵黄抗体的使用。目前使用的疫苗分为种鹅用和雏鹅用的鹅胚和鸭胚化疫苗，这些疫苗在实际生产中发挥巨大作用，因其为全毒苗或弱毒苗，存在着潜伏感染，排毒散毒，毒力返祖的缺陷，基因工程疫苗的研制可解决上述问题。 GPV的三种结构蛋白VP1、VP2、VP3位于同一开放阅读框内，共用同一终止密码子，VP1包含了组成VP2、VP3的所有氨基酸，因其具有这一特殊性，成为本研究的目的基因。本试验参照Genbank中发表的GPV B株基因序列设计并合成了扩增GPV H1株VP1的一对引物，利用PCR技术扩增出长约2.2Kb的目的片段，经酶切鉴定后，将其插入原核表达载体pPRoEXHTb的XbaⅠ、XhoⅠ多克隆位点间，构建了GPVH1株VP1的原核表达载体GP1-pPRO。测序结果表明GPVVP1基因由2199个核苷酸组成，编码732个氨基酸。与B株、YG株进行了同源性比较，核苷酸同源性分别为98.5％，93.18％；氨基酸同源性分别为98.09％，95.77％。同时将该目的基因插入到杆状病毒表达系统的供体质粒pF_ASTB_ACHTb的XbaⅠ、XhoⅠ多克隆位点间，经酶切、PCR鉴定后，将重组的供体质粒GP1-FAST转化到含有杆状病毒和辅助质粒的DH10B_AC感受态细胞中，获得了表达GPV H1株VP1的重组杆状病毒GP1-BAC。 用Antheprot5.0对GPVH1株VP1、VP2、VP3及B、YG株VP1的二级结构、抗原性进行预测及比较，表明GPVH1株VP1、VP2、VP3共同部分的二级结构、抗原性无差别：GPVH1、B、YG株VP1的二级结构、抗原性存在差别。GPVH1株与其他细小病毒VP1的氨基酸序列同源性比较证明GPVH1与依赖病毒AAV-2的同源性高于与自主细小病毒的同源性。 本试验为进一步研究GPV的分子生物学特性，VP1的理化特性、生物学活性奠定了基础：为GPV的诊断试剂、基因工程疫苗的研制做了前期工作：同时为GPV的分类地位及其进化关系提供了佐证。更多还原

【Abstract】 GPV (Goose parvovrius) has been classified by its morPhological, biochemical, andclut-Ure characteristics as a member of the parvovirus genus of the parvoviridae. lt is a highlyfatal disease of gos1ings and Muscovy ducklings, the typical pathological lesions was found indigest tract, specially in small intestine. The disease has broken out in several countries, andalI isolated GPV were similar in antigenicity. The disease was diagnosed mainly ac:cording tolts clinico-pathology and combining the serologica1 and virological methods, but most ofthose methods were time-consuming and laborious, which delay the cure, so it is necessary todevelop a rapid, simple and special diagnostic method. Prevention and cure of the Derzsy’sdisease depended on vaccine and antiserum, antibodies of eggs.The vaccines includes gooseembryo and duck embryo vaccines which were used 1n breed goose and goslings, and thosevaccines have great effect in breeding goose, but the entire virion live vaccines and attenuatedvaccines exist many deficiencies. such as preclinical infection, dissemination of virus,recovery of viru5, etc. Those proplems can be sol\/ed by producing genetic engineering’asclne.GPV contins three capsid protein. VP1. VP2 and VP3.The genes, encoding them, werelocated in a same ORF, and they possess of common stop codons. Furthermore, amino acidsequence of VPl contained the fu1l sequence of VP2 and VP3, so VP1 gene become interestgene. In this research. a pair of primers were designed to amplify VPl gene by PCR accordingto the published sequence of GPV B strain in Genbank. The product of PCR was 2.2 Kb. Afteridentification of PCR product by restriction endonuclease the interest gene was inserted in theTha I. Tho I multiple cloning sites of prokaryotlc expression vector pPRoEXHTb toconstruct the recombinant prokaryotic expression vector GPl -pPRO of VP 1 of GPV strain H 1.The result of sequencing showed that VP1 included 2l99 bases, which encoded,732 amnioacids. The interest gene shared 98.5% and 93. l 8% in bases comparisoning with GPV strains Band YG, and 98.09%, 95.77% homoIogy in amino acid.The interest gene was inserted in the -Tha l. Tho 1 multiple cloning sites of donorplasimd pFAsTBAcHTb of baculovirus expression system. After analysis by restrictionendonuclease and PCR, the recombinant donor plasmid GPl-FAST was transforn1ed to thecompetent celI DHI0BAc which contalns the bacmid and the helper plasmid, the recombinantbacmid GPl -BAC was acquired which would express the VPl of GPV strain H l.The secondary structUre and antigenicity of VP 1, VP2, VP3 of GPV strain H l and VP1s25fot%&&Z Abstract $jhAAk#of GPV strains B, YG were predicted and comparisoned by Antheprot5.0 software. The resultsuggested that there was no difference between VPl, VP2 and VP3 of GPV HIstrain insecondny structure and antigenicity, but VPls of GPV strains Hl, B, YG were different inthose two respects. Comparison of VPl of GPV strain Hl with other parvoviruses showed thatthe homology between GPV strain H1 and AAV-2 was higher than that of GPV stains H1 andother aUtonomous parvoviruses.This study provided the basis for the further research about GPV molecular biologicalproperties and the physical, chemical character and biological activity of VPl. lt was preparedwork for the development of diagnostic reagent and genetic engineering vaccine. And it alsoprovided the evidence for taxonomy of GPV and its relationship in evolution.Postgraduaef Bo Ma Major f Preventive veterinary scienceAdvisor f Pro f Jun wei Wang更多还原