【作者】 何长生；

【导师】 魏建忠；

【作者基本信息】 安徽农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 本研究通过鸡胚接种、动物回归试验、干扰试验、血凝试验和电镜观察等手段分离鉴定了两株肾型IBV（分别命名为AH1-99株、AH2-03株）。在此基础上，为了从分子水平上了解安徽省IBV地方株主要结构基因的遗传变异背景，为防制IBV提供依据，我们利用RT-PCR技术分别扩增出两分离株的S1、S2、M和N基因，并将这些基因插入到pMD18-T载体中构建了重组质粒，随后对这些基因进行了序列测定和序列分析。结果显示：S1基因由1611-1617核苷酸组成，编码537-539aa。在所有结构基因中，S1基因变异程度最大，其核苷酸的变异主要集中在基因的N-端和C－端；同时，AH2-03株在358-363nt处插入了GGGTCT六个碱基。基因内存在一个EcoR I酶切位点和3个Hae III酶切位点。S1蛋白内部存在16－18个潜在的糖基化位点，与M41、Beaudette株S1蛋白相比，分离株S1蛋白内部潜在的糖基化位点数量和位置发生了一些改变，同时其表面抗原性出现了一些变化，如在438－444aa处的抗原位点消失，而在450－457aa处出现了一个抗原性强的位点。在进化树中，两毒株S1基因处在不同的基因群中，其中AH1-99株S1基因同H52、M41、Beaudette等标准毒株以及多数国内分离株在同一个基因群内，与它们同源性为91.7%-92.2%，AH2-03株S1基因同标准毒株亲缘关系较远，而同少数国内分离株在另一个基因群内。与S1基因相比，S2基因其较为保守。S2基因的大小为1881nt，有两个终止密码子，故编码625个氨基酸。基因内部存在着碱基的缺失与插入，两分离株均在895-900nt处插入了GCGACT六个碱基，在1441-1446nt处缺失了ATTACT六个核苷酸。S2蛋白氨基酸序列中有10-11个潜在的糖基化位点，与M41、Beaudette株S2蛋白相比，分离株潜在的糖基化位点数量和位置发生了一些改变。其在第560－600位约40个氨基酸残基高度疏水，为S2蛋白与病毒囊膜结合的区段。两分离株S2基因亲缘关系很近，同源性高达96.2％，并且在S2基因系统进化树中独立形成一个基因群，而与其它参考毒株的同源性只有70.3%-88.9%。比较发现，在四个结构基因中，M基因最为保守，核苷酸同源性为87.2％－95.9％。同多数参考毒株相比较，AH2-03株在16-18nt处插入了GGA三个碱基。M蛋白由225-226个氨基酸残基组成，其内部有两个潜在的糖基化位点。分析发现，AH1-99株M基因和S1基因的进化是平行进行的。虽然两分离株核苷酸有93.4%的相似性，但是在M基因系统发生树中，两者不在同一个基因群内。同S1基因相似，AH1-99株M基因同H120、M41、Beaudette株以及多数国内分离株亲缘关系近，在同一个基因群内；而AH2-03株M基因同上述毒株亲缘关系较远，与少数国内分离株，如SAIB14、GD6-98在另一基因群内。 <WP=4>分离株N基因内无碱基的缺失与插入，同绝大多数IBV毒株一样，其大小为1230nt，编码409个氨基酸，与参考毒株N基因核苷酸同源性为85.2%-97.6%。分析显示，N蛋白大部分由亲水性氨基酸组成，富含碱性氨基酸，这些碱性氨基酸多数成簇集中在63-85aa、198-249aa、330-368aa三个保守的区域内。与M41、Beaudette株S1蛋白相比，分离株N蛋白内部磷酸化化位点的数量和位置发生了一些改变。在IBVN基因进化关系树上，分离株与Beaudette、H52、M41等标准毒株亲缘关系较远，而两毒株之间以及同国内分离株LX4、X等亲缘关系较近。更多还原

【Abstract】 Two field virus in Anhui province were isolated from commercial broiler chickens vaccinated with live Mass viral strain (H120). These isolates were identified by series experiments of biological character involving pathogenicity, dwarfing of chicken embryos, interference of NDV—clone-30 propagation, haemagglutination test and electron microscopy. All facts of experiments proved that the two virus strain is avian infectious bronchitis nephritis virus. Lastly they were designated as AH1-99 strain and AH2-03 strain respectively. In order to trace the origin and evolution of the two field strains, we have obtained the sequences of S1, S2, M and N genes by RT-PCR, cloning, sequencing. Using the software of Dnastar and referencing to others IBV strains’ published sequences in Genbank, we have analyzed molecular characteristics of those structural genes and proteins and constructed phylogenic trees of S1, S2, M, N genes . S1 gene is composed of 1611-1617 nucleotides coding 537-539 amino acids. Sequence analysis revealed that the S1 gene is far more variable than other genes and the highly variable regions locate at the N-terminal and C-terminal of it. Six nucleotides GGGTCT were inserted in the site of 358-363 in AH2-03 strain S1 gene. Interestingly, there is one EcoR I RE site in S1 gene and three Hae III RE sites can be found. In the two strains S1 protein exist 16-18 potential glycosylation sites, and the quantity and location of these potential glycosylation sites varied. Compared with other IBV strains S1 protein, the epitope at 438-444aa disappeared while at 440-457aa presented a strong antigenicity site. According to phylogenic trees of S1 gene, the two isolates fell into different subgroups. AH1-99 strain is closely related to standard IBV strains of H52, M41, Beaudette and most of domestic isolates and the identity within this subgroup is 91.7%-92.2%, while AH2-03 strain is alienated to those standard strains.The size of S2 gene is 1881 nucleotides and the size of S2 protein is 625 amino acids owing to two stop codons in it. S2 is more conservative than S1 gene. Sequence comparison showed that six nucleotides GCGACT inserted at the position of 895-900 and six nucleotides ATTACT deleted at the site of 1441-1446. The pitch of the amino acids locating between 560 and 600 is probably associated with the viral membrane.10-11 potential glycosylation sites present in the S2 proteins of the isolates, and the quantity and location of these potential glycosylation sites changed. A high degree of similarity (96.2%) was observed between the two isolates S2 genes and they formed an alone gene group in <WP=6>S2 gene phylogenic tree. The extent of nucleotides homology between the two field strains and referencing strains is from 70.3% to 88.9%.M protein comprises 225-226 amino acids and two potential glycosylation sites. present in it. Sequence analysis indicated that M gene of the two strains is the most conservative in the four structural genes. Three nucleotides GGA were inserted in the position of 16-18nt in AH2-03 strain M gene. The identity of nucleotides compared with others is between 87.2% and 95.6%. The evolution of AH1-99 strain M gene is parallel to that of S1 gene. In the phylogenic tree of IBV M genes, AH1-99 strain is in the gene cluster that including H120, M41, Beaudette, and majority of domestic field stains. Though AH2-03 strain shared 93.4% consistent with AH1-99 strain, they weren’t in the same gene group. Both AH2-03 strain and a few of inland IBV strains such as SAIB14, GD6-98 were in another subgroup.Sequence analysis suggested that no insertion and deletion of nucleotides in N gene. It contains1230 nucleotides which codes 409 amino acids. The homology of nucleotides with other IBV strains is from 85.2% to 97.6%. Majority of amino acid residues of N protein are hydrophilic. It is abundant with basic amino acids which locate in three conservative regions:63-85aa, 198-249aa, and 330-368aa. Compared with IBV reference strains of M41 and Beaudette, the quantity and location of phosphorylation sit更多还原