【作者】 郁斌；

【导师】 王永坤；

【作者基本信息】 扬州大学 ， 禽病学， 2001， 硕士

【摘要】 利用RT-PCR技术，分别从禽Ⅰ型副粘病毒（APMV-Ⅰ）鸽源分离株（PPMV-Ⅰ）SZb9803株、NC9701株、NP9904株、PK9910株四株中成功扩增出预期大小为约0.5Kb的F基因重要功能区片段；从PPMV-IS Za9803株、YZ9712株、PB9601株三株中分别成功扩增出大小为1.7Kb的F基因，与预期的片段大小相一致，然后进一步将上述四株PPMV-Ⅰ的F基因重要功能区片段和三株PPMV-Ⅰ的F基因分别克隆入pGEM~R-T质粒载体中，进而对所克隆人的片段分别进行鉴定和序列测定及分析。 应用Clustal W软件将上述所得序列与从Genebank和其它文献资料中获取的NDV基因Ⅱ、Ⅲ、Ⅵ、Ⅶ型代表株F基因1-389间的核苷酸序列及推导的氨基酸序列进行同源性分析。结果表明：SZa9803株、SZb9803株、YZ9712株、NC9701株、NP9904株、PB9601株六株PPMV-Ⅰ分离株与新城疫病毒（NDV）广东肇庆鸽源分离株ZQ98-1株的核苷酸、氨基酸同源性分别为：96％-97％、96％-98％，与属于NDV基因Ⅵ型b亚型的英格兰参考株1168/84株、威尔士参考株760/83株的核苷酸、氨基酸同源性分别为：93％-95％、91％-94％；与基因Ⅱ型代表株Lasota株的核苷酸、氨基酸同源性分别为：82％-83％、85％-87％；与基因Ⅲ型代表株F48E8株的核苷酸、氨基酸同源性分别为：84％-85％，85％-87％；与基因Ⅶ型代表株YG97分离株的核苷酸、氨基酸同源性分别为88％-89％、89％-91％。应用PHYLIP软件绘制基因进化树，由进化树可见，SZa9803株、SZb9803株、YZ9712株、NC9701株、NP9904株、PB9601株六株PPMV-Ⅰ分离株均属于NDV基因Ⅵ型的b亚型，且形成一个独立的进化簇。而PK9910株则属于NDV基因Ⅱ型， 依据评价NDV致病性的三个致病指数（最小致死量致死鸡胚平均死亡时间（MDT）、一日龄SPF雏鸡脑内接种致病指数（ICPI）、六周龄SPF雏鸡静脉接种致病指数（IVPI）），我们对五株鸽源分离株的MDT、ICPI、IVPI三个指数分别进行了测定，其结果分别为MDT 87.6-88.8小时，ICPI 1.38-1.53，IVPI 1.00-1.50。为了能够更准确地评价这五株PPMV-Ⅰ分离株对鸽的致病性，我们根据在鸡体上测定IVPⅠ的方法，在五周龄非免疫鸽体上进行了这五株PPMV-Ⅰ分离株的IVPⅠ测定，其结果为：1.78-2.07。而PK9910分离株对鸡和鸽的IVPI值均为0。试验结果表明：SZa9803株、YZ9712株、NC9701株、PB9601株四个鸽源分离株均属于中速型NDV毒株，而对鸽则呈强致病性，相当于速发型NDV毒株。而PK9910分离株属缓发型NDV毒株。 禽副粘病毒I型鸽源分离株融合蛋白基因克隆、测序及致病性的研究2 交叉血凝抑制（HI）试验比较PPW－I分离株与Lasota株结果表明：SZa9803株、SZb9803株、YZ9712株、NC9701株、PB9601株虽与Lasota株有很高的血清交叉反应，对同源血清（抗 SZa9803株血清）的 HI为 1：2’，而与异源血清（抗Lasota株血清）的HI为 1：2’，而PK9910株与抗Lasota株血清的HI为 1：2’，比其它五株叩W－I分离株与抗 Lasota株血清的 HI滴度（1：2‘）高，但己表现出这五株PPMV－I分离株与Lasota株有差异。更多还原

【Abstract】 M. S. Candidate: Yu BinAdvisor: Prof. Wang YongkunAnimal Science And Veterinary Medicine College. Yangzhou UniversityYangzhou, Jiangsu 225009P. R. ChinaABSTRACTAccording to the published Fusion(F) protein gene sequence of Newcastle disease virus(NDV), two pairs of oligonucleotide primers were designed and synthesised. The anticipated nucleotide sequence of F gene function domain with a length of about 500bps were amplified from F gene of pigeon origin paramyxovirus type I (PPMVI )isolates SZb9803.. NC970 1 NP9904~ PK99 10, and the complete gene encoding Fusion protein about 1.7kb were also amplified from F gene of PPMV- I isolates SZa98O3 ~. YZ9712~ PB9601 by reverse transcription-polymerase chain reaction (RTPCR).Four fragments with a length of about 500bps and three fragments with a length of about 1.7kb were all cloned into plasmid vector pGEMR~T respectively and further sequenced.Compared with those of NDV genotype IL. IlL. VL VII reference strains, the nucleotide sequences from nucleotide 1-389 in F gene encoding region and deduced amino acid were aligned and analysed by software Clustal W, the result indicate that those of six PPMV- I isolates and Zhaoqing isolate ZQ98-l of pigeon origin had 96%97%.. 96%-98% homology in nucleotide and amino acid level respectively, The six PPMV- I isolates above and the England reference strain 1168/84.. Wales reference strain 760/83 of PPMV- I had 93%-95%.. 91%-94% homology in nucleotide and amino acid level respectively. Also the six PPMV- I isolates above and strain Lasota of NDV genotype II -. strain F4~E~ of NDV genotype III-. isolate YG97 of NDV genotype VII had 82%-83%~. 84%-85%.. 88%-89% homology in nucleotide level and 85%-87%~.485%-87% ~. 89%-91% homology in amino acid level respectively. Based on the sequences from Genebank, the phylogenetic tree was constructed using software PHYLIP, and it showed that the cluster in NDV genotype VI including six isolates was formed, and isolate PK99 10 belonging to NDV genotype II, clearly distinct from all other PPMV- I isolates.According to the three incides of measuring the pathogenicity of NDV: the mean death time(MDT)~. the intracerebral pathogenicity index(ICPI) with 1-day-old SPF chickens the intravenous pathogenicity index(IVPI) with 6-week-old SPF chicken, they were measured for five PPMV- I isolates, and the results were that the MDT of five isolates was 87.6-88.8 hours, the ICPI was 1.38-1.53, the IVPI with 6-week-old SPF chicken was 1.00-1.50. The five isolates were also measured using five-week-old pigeon, and the IVPI was 1.78-2.07. The IVPIs of isolate PK9910 measured using chicken and pigeon all were 0. These results confirmed the five PPMV- I isolates measured were mesogenic NDV to chicken and velogenic NDV to pigeon.The difference of the PPMV- I isolate and strain Lasota compared by crossreaction of hemagglutination inhibition (HI)test, and showed that the PPMV- I isolates, except PK99 10, had some difference with strain Lasota, although they all had high level of cross-reaction to Lasota in HI test.更多还原