含甲型H1N1流感病毒HA基因序列病毒样颗粒的制备  

CONSTRUCTION OF THE VIRUS-LIKE PARTICLES CONTAINING HA GENE FRGMENTS WITH TYPE A INFLUENZ A(H1N1)VIRUS

作　　者：单云峰[1] 戚宇华[1] 单军[1] 葛以跃[1] 赵康辰[1] 崔仑标[1] 史智扬[1] 汪华[1] 

机构地区：[1]卫生部肠道病原微生物重点实验室,江苏省疾病预防控制中心病原微生物研究所,南京210009

出　　处：《现代预防医学》2011年第22期4714-4716,4719,共4页Modern Preventive Medicine

摘　　要：[目的]构建耐RNase酶的内含甲型H1N1流感病毒HA基因序列的病毒样颗粒。[方法]构建中间载体pET32a-MS2,将甲型H1N1流感病毒的HA基因片段连接中间载体上,构建原核表达载体pET32a-MS2-HA,转化宿主菌,诱导表达制备病毒样颗粒,对病毒样颗粒进行荧光定量RT-PCR检测和稳定性实验。[结果]表达载体经PCR、酶切鉴定分析后证实构建成功,荧光定量RT-PCR实验表明该病毒颗粒含有HA基因片段并且颗粒稳定性良好。[结论]成功构建了含甲型H1N1流感病毒HA基因序列的病毒样颗粒且稳定性良好,有望作为甲型H1N1流感病毒RNA检测的标准品和质控品。[Objective]To construct RNase-resistant virus-like particles containing HA gene sequences of type A influenza（H1N1）virus that can be used as RNA standards and controls in RNA virus detection.[Methods]The coat protein and maturase gene of E.coli bacteriophage MS2 was amplified by PCR,then the gene was cloned into pET32a to construct the intermediate vector pET32a-MS2.The hemagglutinin gene（HA）sequence of type A influenza（H1N1）virus was cloned into the downstream of pET32a-MS2 bacteriophage gene to construct the prokaryotic expression vector pET32a-MS2-HA.The recombinant plasmid were transformed into E.coli cells,and then induced by IPTG.The expression products were identified by RT-PCR.Then the virus-like particles were tested by real-time fluorescence quantitative RT-PCR and stability experiments.[Results]The results of real-time fluorescence quantitative RT-PCR and stability test showed that the virus-like particles contained the HA gene fragment of H1N1 and had good stability.[Conclusion]As a RNase-resistant virus-like particle,pET32a-MS2-HA,has good stability and can be used as RNA standards and quality controls for influenza A H1N1 virus detection.

关 键 词：MS2噬菌体 甲型H1N1流感病毒 病毒样颗粒 原核表达 

分 类 号：R373.13[医药卫生—病原生物学]