肾型传染性支气管炎病毒的鉴定和结构蛋白的表达

Characterization of a nephropathogenic infectious bronchitis virus strain and expression of its structural proteins

对分离自江苏省某发病蛋鸡群中1株病毒(Ck/Jiangsu/DS10/2008,DS10)进行了生物学特性鉴定。同时利用RT-PCR扩增该病毒的S基因、M基因和E基因并进行测序,与GenBank中其他参考株构建进化树,进行比对分析,研究其遗传进化关系。另将S基因、M基因和E基因克隆至杆状病毒表达系统pFastBac 1载体并转染Sf9细胞进行表达,用间接免疫荧光鉴定其表达,用琼扩试验验证表达产物的反应性。经生物学特性鉴定,DS10病毒为嗜肾型传染性支气管炎病毒(IBV)。遗传进化分析表明,各基因之间的遗传进化关系无明显相关性。间接免疫荧光检测表明,S蛋白、M蛋白和E蛋白在Sf9细胞中得到表达。琼扩试验证实,S基因和M基因的表达产物具有良好的反应性,而E蛋白未发现与阳性血清反应所产生的特异沉淀线,表明所表达的S蛋白和M蛋白具有良好的生物学活性,E蛋白可能因其自身分子质量小,在病毒中的含量较少相关。研究结果提示,现有传染性支气管炎疫苗对流行株保护效果不佳,表达的S蛋白。

This research was conducted to study an isolate of infectious bronchitis virus（Ck/Jiangsu/DS10/2008,DS10） from layer poultry farm in Jiangsu province and to provide information for the control of the disease.DS10 virus isloate was characterized by a series of biological test.The S,M and E genes of DS10 virus were amplified by reverse transcription polymerase chain reaction（RT-PCR）.The amplified fragments were sequenced and phylogenic tree of the genes were constructed with genes of other viruses from GenBank.Subsequently,these genes were cloned into the pFastBac 1 vector of Bac-to-Bac baculovirus expression system and transfected into Sf9 cell line with the expression and biological activity detected using indirect immunofluorescentassay（IFA） and agar immunodiffusion,respectively.Phylogenetic analysis showed that there was no significantly correlation between different genes of DS10 virus.DS10 virus was identified as nephropathogenic strains by its biological properties.Indirect fluorescence assay（IFA） showed that S,M and E proteins were expressed in Sf9 cell line.The agar immunodiffusion assay showed that S and M proteins expressed in Sf9 cell had positive precipitation band with serum against IBV,while the E protein did not being found precipitation.Results indicated that the expressed S and M proteins had powerful biological activity.The low rate in virus total protein and small molecular weight of E protein may lead to invisible of precipitation band in neutralization with serum.For the vaccine strain of IBV at present can not provid full protection against to variant viruses challenge,the expressed proteins of S,M and E in the present study afford fundamental data for the research of its biological activity and development of diagnosis antigen and subunit vaccine.