表达H5亚型禽流感病毒不同形式HA基因重组鸭瘟病毒的构建及HA表达形式的鉴定  

Construction and expression of recombinants duck enteritis virus expressing different forms of hemagglutinin from H5 subtypes avian influenza virus

作　　者：刘璐[1] 丁蕾蕾[2] 柳金雄[2] 陈普成[2] 李爱心[2] 胡玉珍[2] 姜永萍[2] 陈化兰[1,2] 孙晓林[1] 

机构地区：[1]甘肃农业大学动物医学学院,甘肃兰州730070 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部动物流感重点开放实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2016年第4期266-270,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31302064)

摘　　要：为研究禽流感病毒(AIV)血凝素(HA)基因的信号肽以及跨膜区对HA蛋白在重组病毒中表达的影响,本研究以鸭瘟病毒(DEV)为载体,在DEV基因组的US7和US8基因之间通过同源重组插入去除信号肽或跨膜区的H5N1亚型AIV HA基因编码序列,分别构建了缺失信号肽HA基因的DEV重组病毒(r DEV-PK)和缺失跨膜区HA基因的DEV重组病毒(r DEV-PSM),并传至20代,经PCR对插入的外源基因编码序列扩增及序列测定,表明外源基因能够在重组病毒基因组中稳定存在;间接免疫荧光和western blot鉴定结果表明去除信号肽的HA蛋白能够在重组病毒感染的鸡胚成纤维细胞浆中正确表达,而去除跨膜区的HA蛋白能够在感染细胞的培养液中检测到。本研究为表达H5N1亚型AIV HA基因重组DEV载体弱毒疫苗在鸭体内免疫机制的进一步研究奠定基础。To evaluate the signal peptide and transmembrane region effects on the expression of hemagglutinin（HA） protein of avian influenza virus（AIV） from recombinant viruses, we constructed and rescued two duck enteritis viruses（DEV）recombinant viruses by use of homologous recombination to insert the signal peptide deleted（r DEV-PK） and transmembrane region deleted（r DEV-PSM） HA gene of the H5N1 subtype AIV HB49（H5N1） strain between the US7 and US8 genes of the DEV genome, respectively. Both of recombinant viruses were serially passaged for 20 passages in CEF cells. The identified results indicated that both forms of HA were stably existed in the recombinant viruses detected by PCR and sequencing. The indirect immunofluorescence assay and western blot results showed that the HA proteins were only detected in the cytoplasm of CEF infected with r DEV-PK. However, the HA proteins were detected in the cell culture medium of cells infected with r DEV-PSM. In conclusion, the transmembrane region deleted HA protein was able to effectively secrete from the r DEV-PSM infected cells. This study provided the basis for further immune protection test and the immunologic mechanism in duck induced by recombinant DEV-vectored live vaccine expressing the HA gene of H5N1 subtype AIV.

关 键 词：禽流感病毒 HA蛋白 重组鸭瘟病毒 信号肽 跨膜区 

分 类 号：S852.65[农业科学—基础兽医学]