【作者】 马德星；

【导师】 李广兴；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 本研究从我国四川省一疑似鸡传染性支气管炎（Avian infectious bronchitis virus, IB）的雏鸡病料中成功分离到一株鸡传染性支气管炎病毒（Infectious Bronchitis Virus, IBV）（命名为SC）。病毒经鸡胚传代、血凝实验监测和负染电镜检查，证实为IBV。采用自行设计的引物经反转录聚合酶链式反应（Reverse-translation polymerase-chain reaction, RT-PCR）技术扩增得到了SC株、HN株（河南分离株）mRNA 5 cDNA（可编码重叠的5a、5b两个蛋白）及SC、HN、HLJ（黑龙江分离株）、SD（山东分离株）、AH（安徽分离株）五株IBV mRNA 6 cDNA（编码N蛋白）。应用DNAstar 5.06，Clustal 1.8，Antheprot 5.0分析软件将克隆测序的5a，5b蛋白基因和N蛋白基因分别与GeneBank中14株国内外参考毒株进行序列比较分析和基因同源性分析，绘制了5a、5b和N基因的进化树，同时描述了核蛋白的抗原表位、疏水性等基本特性。结果如下：1. 对5a基因核苷酸、氨基酸进行了序列同源性和系统进化树分析，结果表明：SC、HN两毒株间有较近的亲缘关系，在同一进化群内，两毒株间5a基因核苷酸和氨基酸的同源性均较高；两毒株与国内外参考毒株相比存在多处独特的点突变，导致相应区域的氨基酸发生改变；在5a基因核苷酸及推导的氨基酸同源性上，SC株、HN株与H52疫苗株较高，推测5a基因的遗传变异与该疫苗株的应用可能有一定关系。2. 对5b基因核苷酸、氨基酸序列同源性分析表明：在5b基因核苷酸及氨基酸同源性上，HN株与SC株间最高；HN和SC株5b基因核苷酸及推导的氨基酸同源性均明显高于5a基因，其它参考毒株的5a和5b序列分析亦如此，说明5b基因的保守性要高于5a基因；对5 b基因进化树分析表明，SC株、HN株亲缘关系较近，进化过程中发生的变异可能相似。3. 从序列同源性、进化树及二级结构预测三个方面分析了相对保守的N基因的遗传变异，结果表明：HN、SC、AH三个分离株N基因的变异相似，SD、HLJ两个分离株的变异相似；HN、SC、AH三个分离株的变异独特，明显不同于国内外参考毒株，推测该三个毒株具有共同的起源，但其确切的起源还需要分析最能反映IBV田间进化的S1基因的变异情况；HN、SC、AH株三个分离株在起源相同，进化相似的可能情况下在病毒与宿主、病毒与环境的相互作用等选择压力下进行着不同程度的衍化而在基因的局部区域具备了各自的特征。综上所述，本文分析了HN、SC两株IBV 5a、5b基因和五株IBV N基因的遗传变异，这将为进一步揭示国内不同IBV毒株的遗传演化关系、IBV的分子流行病学以及IBV基因工程疫苗的下游研究工作提供重要的理论依据。更多还原

【Abstract】 In this study,we successfully isolated an infectious bronchitis virus (IBV) strain (named SC isolate) from a chicken flock with typical clinical symptom of infectious bronchitis (IB) in Sichuan Province in China. The virus was passaged in chicken embryos, characterized by Haemagglutination (HA) experiment and detected by electron microscope. cDNA of mRNA5 (encoded two overlap protein 5a, 5b) gene of IBV SC and HN strains, mRNA6 (encoded N protein) gene of IBV SC, HN and AH strain (isolated in Anhui Province), SD strain (isolated in Sandong Province) and HLJ strain (isolated in Heilongjiagn Province) were amplified according to designed specific primers by reverse transcription polymerase chain Reaction (RT-PCR) from viral RNAs. Compared the 5a, 5b gene and N gene respectively with the other 14 IBV strains from GeneBank, homology of nucleotide and deduced amino acid sequence were analysed by DNAstar software. Phylogenetic tree was mapped with N gene, 5a gene and 5b gene. Hydrophobility and antigen epitop were described.These results were as follows:1. Analysis of homology and phylogenetic tree of 5a gene indicated that HN and SC isolate had closer relationship and were classified as the same phylogenetic group. HN strain shared high homology with SC isolate in nucleotide and deduced amino acid sequence. The genetic variation of HN and SC isolate differed from other strains because of existence of some specific point mutations. The genetic variation in 5a gene of SC isolate was more obvious compared with HN isolate. The 5a gene of HN and SC strain shared high homology with gene 5a of H52 strain. This indicated that the evolution of 5a gene of the two virus isolate was influnced by application of vaccine strain. 2. Analysis of 5b gene showed that the homology of nucleotide and deduced amino acid sequence of HN and SC isolate were all the highest compared with those of other strains. The homology of nucleotide and deduced amino acid sequence of 5b gene of HN and SC isolate was obviously higher than that of 5a gene. So was the analysis of 5a, 5b gene of other reference strains. This indicated that the 5b gene was more conservative than 5a gene. The phylogenetic tree analysis of 5b gene of HN and SC isolate showed that the two isolates had closer genetic relationship. The genetic variation of 5b gene of SC and HN isolate was similar to each other. 3. Through annlysis of homology, phylogetic tree and predicted secondary structure of relative conservative N gene, we observed that the genetic variation was similar among N <WP=10>gene of SC, HN and AH isolate. The genetic variation of these three isolate was different from all the freign strains and other China strains. The three isolates, HN, AH and SC isolate, had the same original and experienced the same evolution process in vicinity areas, but not originated from vaccine strain. So was the HLJ, SD strain. The genetic analysis of S1 gene will help us to interpret the origin of HN, SC and AH isolates. The strains, isolated in different area of China, perhaps experienced different evolution under the condition of different time and space, but in the same area, the isolate was influnced by complicated selected pressur which led to the different genetic characterization in local gene region.In conclusion, the genetic variation of non-structural proteins genes 5a, 5b of HN and SC strains, N gene of HN, SC, AH, SD, HLJ isolate were analysed in this paper. It would be useful for the study of molecular epidemiology of IBV and help us to study the evolution relationships of different IBV isolates and develop new IBV vaccines.Candidate:Ma DexingMajor:Basic Veterinary ScienceSupervisor:Prof. Li Guangxing更多还原