含有H3N8马流感病毒HA基因噬菌体病毒样颗粒的构建与纯化  

Construction and purification of virus-like particles containing HA gene of H3N8 equine influenza virus

作　　者：张晓文 姜帆 孙明君 郑小龙 于业峰 王群 

机构地区：[1]山东出入境检验检疫技术中心,山东青岛266002 [2]青岛检验检疫技术发展中心,山东青岛266002

出　　处：《中国预防兽医学报》2015年第3期194-198,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：山东出入境检验检疫局科技项目(SK201201);国家科技支撑计划项目(2012BAK11B00)

摘　　要：为构建含H3N8马流感(EIV)HA基因的噬菌体病毒样颗粒(VLPs),本研究将编码组氨酸标签序列插入MS2噬菌体编码外壳蛋白的β发夹环结构序列中,构建含有重组组氨酸标签的MS2噬菌体外壳蛋白和表达成熟酶蛋白基因的重组质粒p NH-MS2his。并通过RT-RCR技术扩增H3N8亚型EIV的HA基因,将其克隆于p NH-MS2his中,构建原核重组表达质粒p NH-MS2his-H3,并将其转化于大肠杆菌BL21(DE3)中进行诱导表达。结果表明,表达的重组蛋白能够自主装配形成VLPs,其中含有H3N8亚型EIV的HA基因RNA序列,并且该RNA序列具有良好的稳定性,可以作为RNA病毒检测时的标准品和质控品用于临床及海关样品检测。The aim of this study is to construct virus-like particles （VLPs） containing HA gene of H3N8 equine influenza virus （EIV） based on the RNA-binding protein of bacteriophagic MS2 coat protein. The universal expressing vector pNH-MS2his was successfully constructed by inserting histidine to β-ring of MS2 bacteriophage coat protein. Then, HA gene of EIV was amplified by RT-PCR and cloned into vector pNH-MS2his to construct the prokaryotic expression recombinant plasmid of pNH-MS2his-H3 and transformed into E.coli BL21 （DE3） for expression. The expressed recombinant protein automatically refolded to form the VLPs, and the RNA of HA gene in the VLPs were identified by PCR and RT-PCR. The stability of VLPs was tested by real-time RT-PCR and the results showed that the RNA was packaged in the VLPs. The VLPs can be used as a standard and quality control in RNA virus detection

关 键 词：H3N8亚型马流感病毒 假病毒颗粒 Armored RNA技术 

分 类 号：S852.65[农业科学—基础兽医学]