【作者】 孟凡磊；

【导师】 刁有祥；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 鸡传染性支气管炎(Infectious Bronchitis,IB)是由传染性支气管炎病毒(Infectious bronchitis virus,IBV)引起的一种急性、高度接触性传染病。自30年代在美国爆发以来,现已成为世界各地流行的重要禽病。IBV是单股RNA病毒,该病毒基因可因点突变和重组而发生变异,故传染性支气管炎病毒血清型较多。已知的血清型有以侵害呼吸道为主的Conn、Iowa97、JMK、Florida、Arkansas99等和以侵害肾脏为主的M41、Holte、Gray、Australia“T”等30余种。Gough等(Gough et al,1992)报道了英国一种新的793/B血清型的IBV,该毒株和其它血清型传支毒株之间无交叉血清学关系;其免疫原基因S1的序列与欧洲17个传支毒株之间差异高达21～25%。2003年刁有祥、杨杰华等从山东省某蛋鸡饲养场产蛋异常下降鸡群中分离到一株793/B传染性支气管炎毒株,命名为TA03株。目前,该血清型IBV在西班牙、德国、荷兰、中国、意大利、泰国等国均有发生和流行,对养鸡业危害较大。根据GenBank中已经发表的IBV N基因的保守序列,设计合成一对引物,利用RT-PCR技术扩增IBV N基因的582 bp的核酸片段,并制备出地高辛标记的IBV核酸探针。特异性检测结果表明,该探针能与不同毒株的IBV核酸发生特异性杂交,而与对照的NDV、鹅副粘病毒的核酸杂交反应为阴性;敏感性检测结果表明,该探针对IBV的最低检出量为10 pg,显示所制备的核酸探针用于IBV的检测是可行的。参照GenBank已经发表的793/B和多株IBV的N基因和S1基因,设计两对引物,对样品的核酸模板进行扩增,建立了一种能够同时检测鸡传染性支气管炎病毒和鉴定出793/B血清型的双重RT-PCR方法。该方法对793/B血清型可检测到582bp和891bp两条核酸片断,其他的血清型只出现582bp一条核酸片断,而新城疫病毒、传染性喉气管炎、鹅副粘病毒菌未检测到特异型条带。该方法简化了聚合酶链式反应程序和缩短了检测时间。根据Genbank已经发表的传染性支气管炎病毒(IBV)N全基因组序列设计引物,对IBV 793/B分离毒株N基因进行克隆与序列分析。结果表明,IBV 793/B的N基因由1229bp组成,与Genbank已发表的11株IBV的N基因相比较,IBV 793/B的N基因共有88处点突变,在第991位发生了一个核苷酸的缺失。N基因的核苷酸同源性为86.9～91.4%,氨基酸同源性为75.8～77.5%。表明IBV 93/B的N基因存在着较大的变异性。更多还原

【Abstract】 Infectious Bronchitis(IB) is a severe acute respiratory disease of poultry, caused by Infectious bronchitis virus (IBV)。Since broke out in USA in the 1930s, IB have been an important disease of poultry around the world. IBV is a sRNA virus , the gene of the virus always mutated owing to it’s point-mutation and gene recombination, so the virus have many serotypes. The known serotypes can be classified into respiratory symptomatic serotypes such as Conn, Iowa97, JMK, Florida, Arkansas99 and nephritic symptomatic serotypes such as M41, Holte, Gray, Australia‘T’.Gough reported a new serotype of IBV named 793/B (also known as 4/91 and CR88) in UK in 1992,793/B isolates have a little serological relation with other serotypes of IBV, the whole S1 gene of 793/B isolates differed by 21% to 25% from those of other 17 European isolates. Youxiang Diao, Jiehua Yang et.al.(2003) isolated an IBV 793/B strain named IBV TA03 from layer flock in a chicken house of Shandong province. In recent years, the 793/B serotype of IBV, harmful to the healthy development of poultry breeding, prevailed and spread in Spain, Germany, Holland,China ,Italy and Thailand, too.According to the genomic sequences of N gene of infectious bronchitis virus (IBV) published in Genbank,one pairs of primers were designed for amplifying the 582 bp fragment in RT-PCR experiments. The PCR product was labeled with digoxigenin as DNA probe for detection of IBV. The hybridization assay result of specificity showed all RNA of IBV strains were positive, but other nucleotide extracted from NDV, GPV were negative. The sensitivity result showed that as few as 10 pg RNA amount of IBV could be detected by DIG-labeled probe. So the DIG-labeled probe could be used to detect the IBV.A Multiplex RT-PCR was optimized to simultaneously detect IBV and 793/B,for saving time of detection. Two sets of primers were designed according to the sequences of 793/B and other IBV at the GenBank. The product of 582bp and 891bp were generated only with RNA from 793/B, where as 582bp product only with RNA from other IBV. However, the RT-PCR failed to detect NDV, ILTV and GPV. The results showed that the established Multiplex RT-PCR technique provided a more sensitive method for diagnosis and epizootic study of the IBV.According to the genomic sequences of Nucleocapsid gene of IBV published in Genbank , a pair of primers were designed. The nucleocapsid protein(N) gene was amplified by RT-PCR. The results of sequencing showed that the complete genome of nucleocapsid of IBV isolate (793/B) consisted of 1229 nucleotides. Sequence analysis showed that 88 point mutations were found in N gene, and one nucleotides was deletion in 991 position. The homology of nucleotide sequence were 86.9—91.4%, and the homology of deduced aminoacids were 75.8 -77.5% compared with the 11 strains published in GenBank. The results showed that these were major variance in the N gene of 793/B.更多还原