【作者】 高宏丽；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 鹅副粘病毒（GPMV）是副粘病毒科，副粘病毒亚科，腮腺炎病毒属，APMV-1中的成员，引起鹅副粘病毒病。该病自1997年由扬州大学王永坤教授报道以来，在国内很多地区的鹅群中流行。鹅副粘病毒病是以消化道病变为主要特征的具有高度发病率和死亡率的烈性传染病，给这些地区的养鹅业造成了巨大的经济损失。初步实验表明，GPMV与传统的NDV的致病性不同，并且常规的NDV疫苗不能提供有效的保护。因此从分子生物学水平上对该病原进行研究具有重要的意义。血凝素-神经氨酸酶（HN）糖蛋白和融合（F）糖蛋白是GPMV的两种主要的囊膜糖蛋白。HN糖蛋白具有血凝素和神经氨酸酶两种活性，F糖蛋白的裂解能力是病毒毒力的主要决定因素。本研究根据GenBank发表的GPMVSFO2株及其它NDV全基因序列，借助Oligo4.1软件，分别设计两对和一对引物，分别采用RT-巢式PCR和RT-PCR方法对鹅副粘病毒JS/1/97/Go株的HN基因和F基因进行了体外扩增，扩增出与预期设计大小相符的HN基因和F基因的特异性条带。将扩增出的DNA片段克隆到pMD18-T载体中，转化TG1感受态细胞，经AMP/IPTG/X-gal平板筛选，酶切和PCR鉴定后获得阳性重组质粒。序列测定表明：HN基因全长1716bp，编码571个氨基酸残基，与参考序列比较，核苷酸的同源性为82.1%-98.4%, 推导氨基酸同源性为88.3%-99.5%,有4个糖基化位点,其中3个高度保守。F基因全长为1662bp，编码553个氨基酸残基,与参考序列相比,核苷酸的同源性为84.2%-99.8%,氨基酸同源性为87.7%-99.8%,有6个糖基化位点,其中5个高度保守。F蛋白裂解位点的氨基酸顺序为112R-R-Q-K-R-F117，与NDV的强毒株特征相符。根据测序结果,利用DNAstar软件预测抗原表位,参考测得的HN基因的序列设计一条引物，扩增HN基因5’端的含有三个抗原表位的基因片段(命名为HNp),测序结果表明长为606bp,编码201个氨基酸残基,与HN全长基因的相应区域同源性比较,核苷酸和氨基酸残基各有2个变异，但不改变表位预测结果。将HN基因和HNp基因亚克隆到原核表达载体pProEX HTb中，将鉴定为阳性的重组质粒转化到DH5α中。将F基因分别亚克隆到原核表达载体pGEX-6P-1和原核表达载体pET-30a(+)中，将鉴定为阳性的重组质粒转化到TG1中。本实验克隆了JS/1/97/Go株的HN基因和F基因，根据测序结果进行序列分析，并与参考的NDV毒株进行了核苷酸和推导氨基酸的同源性比较，为证实GPMV和传统NDV之间的亲缘关系提供了理论基础，同时预测了F和HN基因的抗原表位，构建了含有GPMV的F基因、HN基因、HNp基因的原核表达载体，为原核表达和进一步研究GPMV的致病性及鉴别诊断提供了分子生物学水平上的理论依据，并为表位疫苗的研究奠定了基础。更多还原

【Abstract】 Goose paramyxovirus disease is an acute contagious disease caused by GPMV, a member of Rubulavirus family. GPMV has been found in many areas since it was reported by Wang Yongkun of Yangzhou university in 1997, which was characterized by the lesion of digestive tract with high mortality and occurrence, and has caused a large loss in economy. The primary study show that there is a difference in pathogenicity between GPMV and traditional NDV, and the common NDV vaccine doesn’t provide an effective protection against disease. So it is of great interest to study the pathogen from the molecular biology.HN(Hemagglutinin-Neuraminidase) protein and F(Fusion) protein are two important membrane glycosylated proteins. HN protein has hemagglutinin activity and neursminidase activity. The cleavage site of F protein is the mainly decisive factor in virulence. In this study, according to the complete genome sequence of GMPV SFO2 isolate and other NDV strains published in GenBank, two pairs of primers were designed to amplify HN gene by RT-nested PCR, and one pair of primers were designed to amplify F gene by PCR. The specific DNA products were cloned into PMD18-T vector, and transformed into competent cell TG1, and the positive recombinant plamids were screened by AMP/IPTG/X-gal ,and endonuclease digesting and PCR identification. The sequence analysis showed that HN gene is 1776bp in length, encoding 571 amino acid residues; the homology of nucleotide between JS/1/97/Go strain and other strains varies from 82.1%-98.4%, and the homology of the deduced amino acid varies from88.3%-99.5%; the F gene is 1662bp in length, encoding 553 amino acid residues, the homology of nucleotide varies from 84.2%-99.8%, and the homology of the deduced amino acid varies from 87.7%-99.8%. The amino acid duduced from HN gene has four glycosylated sites, three among them are very conservative. The amino acid duduced from F gene has six glycosylated sites, five among them are very conservative. The amino acid sequence of cleavage site is 112R-R-Q-K-R-F117 according with the virulent NDV strain. The epitopes were predited by DNAstar software according to the sequencing results of HN and F. A single primer was designed to amplify the 5’terminus of HN gene(named as HNp)which includes three epitopes. The sequencing result of HNp gene show there is only two variations in nucleotide and deduced amino acid residues <WP=10>respectively. The HN gene and HNp gene were subcloned into prokaryotic expression vector pProEXHTb respectively ,then were transformed into competent cell DH5α, the positive colony containing recombinant plamsid were screened by extracting plasmid and endonuclease digesting and PCR identification. The F gene were subcloned into prokaryotic expression vector pET-30a(+) and pGEX-6P-1 respectively, then was transformed into competent cell TG1 and screen positive colony.In this research, HN gene and HNp gene and F gene were cloned and analyzed sequence, and their homologous comparision with NDV were done. According to the sequencing results ,the epitopes of HN gene and F gene were predicted ,providing a theoretical foundation for studying the relationship between GPMV and NDV. Three kinds of prokaryotic expression vector containing HN gene, HNp gene and F gene were constructed respectively, laying a base for expessing and further differentiating diagnosis and developing epitope vaccine .Candidate:Gao HongliMajor:Preventive Veterinary ScienceSupervisor:Prof.Wang Junwei更多还原