甲3型流感病毒M基因披甲RNA的研制  

Preparation of armored RNA containing M gene of influenza H3N2

作　　者：于新芬[1] 潘劲草[1] 黄志成[1] 叶榕[1] 寇宇[1] 

机构地区：[1]杭州市疾病预防控制中心微生物检验科,310006

出　　处：《中华实验和临床病毒学杂志》2007年第4期343-345,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：杭州市医药卫生科技项目(2005A009)

摘　　要：目的研制内含甲3型流感病毒M基因RNA的披甲RNA。方法将MS2噬菌体基因组中编码成熟酶蛋白、包膜蛋白和pac位点的DNA片段克隆到表达质粒pET30b中,构建重组质粒pAR-1。将甲3型流感病毒M基因连接到pAR-1pac位点的下游,诱导表达出内含M基因RNA的披甲RNAAR-2,并进行纯化、定量和稳定性研究。结果成功构建和表达了耐核糖核酸酶、内含M基因RNA的披甲RNA,每1mlLB培养基可诱导表达出约8.9×1011个拷贝的AR-2。结论制备的AR-2稳定,无生物传染危险性,可作为甲型流感病毒M基因核酸检测的标准品和质控品。Objective To prepare the armored RNA containing M gene of influenza H3N2, Methods The vector pAR-1 was constructed from expression vector pET30b in which the bacteriophage MS2 DNA fragment, containing the genes for maturase and coat protein and the pac site. was inserted. The M gene fragment of influenza A was inserted into the Hind m site downstream of the pac site on the pAR-1, which formed a new recombinant plasmid pAR-2. After the prokaryotic expression was carried out, armored RNA AR-2 containing M gene was obtained, AR-2 was purified, and then was quantified by real time RT-PCR. Moreover, the stability of AR-2 was checked, Results AR-2 was expressed successfully. AR-2 remained stable under various storage environments, Approximately 8.9 × 10^11 copies of AR-2 particles can be purified from one milliliter of culture. Conclusion It showed that AR-2 was stable and RNase-resistant, which, as a virus surrogate, would be used as RT-PCR standards, controls and training or proficiency samples.

关 键 词：流感病毒A型 RNA 质量控制 原核细胞 表达的序列标记 

分 类 号：R346[医药卫生—基础医学]