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.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
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)
关键词
禽流感病毒
HA蛋白
重组鸭瘟病毒
信号肽
跨膜区
avian influenza virus
HA protein
recombinant DEV
signal peptide
transmembrane region
