含H5N1禽流感病毒核酸序列的病毒样颗粒的制备及应用  

Preparation of the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 and its application

作　　者：戚宇华[1] 崔仑标[1] 史智扬[1] 葛以跃[1] 李显[1] 张文帅[1] 单军[1] 汪华[1] 

机构地区：[1]卫生部肠道病毒重点实验室,江苏省疾病预防与控制中心检验科,南京210009

出　　处：《中国人兽共患病学报》2010年第1期29-32,35,共5页Chinese Journal of Zoonoses

基　　金：江苏省国际科技合作项目(BZ2008068)

摘　　要：目的构建耐RNase酶的内含禽流感病毒H5N1亚型部分核酸序列的病毒样颗粒。方法构建中间载体pET32a-MS2,将禽流感病毒H5N1亚型的HA、NA和M基因片断连接到中间载体上,构建原核表达载体pET32a-MS2-HA,pET32a-MS2-NA和pET32a-MS2-M,分别转化宿主菌,诱导表达制备病毒样颗粒。结果表达载体经PCR、酶切鉴定和测序分析后证实构建成功。电镜观察到了病毒样颗粒,荧光定量RT-PCR试验表明颗粒稳定性良好。结论成功构建了含禽流感病毒H5N1部分核酸序列的病毒样颗粒且稳定性良好,可作为禽流感H5N1亚型RNA检测的标准品和质控品。To prepare the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 for use as RNA standard and control in RNA virus detection,the genes coding the coat protein and maturase of E.coli bacteriophage MS2 were amplified by PCR and then cloned into prokaryotic expression vector pET32a to construct the intermediate vector pET32a-MS2.In addition,the gene sequences coding hemagglutinin（HA）,neuraminidase（NA）and M protein of the H5N1 virus were also cloned separately to the down-stream of plasmid pET32a-MS2,thus constructing the prokaryotic expression vectors pET32a-NS2-HA,pET32a-MS2-NA and pET32a-MS2-M.These recombinant plasmids were then transformed separately to E.coli BL21（DE3）with induction by IPTG.to express the virus-like particles.The virus-like particles observed under electron microscopy were identified by RT-PCR,while their stability was confirmed by real-time RT-PCR.In this way,the virus-like particles were successively constructed and identified through PCR amplification,enzymolysis identification and sequencing analysis.These virus-like particles observed under electron microscopy appeared to be circular in shape with a diameter of about 50 nm.Their stability was proved to be rather good.From these observations,it is apparent that these virus-like particles can be used as RNA standard and quality control in the detection of avian influenza virus H5N1.

关 键 词：MS2噬菌体 禽流感病毒H5N1 病毒样颗粒 原核表达 

分 类 号：R184.3[医药卫生—流行病学] R373[医药卫生—公共卫生与预防医学]