禽流感病毒H5N1抗原基因克隆及体外转录

Cloning of HA,NA and M gene of H5N1 avian influenza virus and preparation of RNA fragments with transcription in vitro

目的通过基因克隆和体外转录,获得H5N1禽流感病毒抗原基因的RNA全长片段,为病原学检测提供阳性定量标准品。方法设计H5N1禽流感病毒血凝素(HA)、神经氨酸酶(NA)及基质蛋白M(M)的基因克隆引物,RT-PCR从病毒RNA获得相应片段,分别连接至pGEM-T Easy质粒并筛选阳性重组质粒,测序鉴定后酶切线性化,用T7 RNA聚合酶进行体外转录,产物用DNase酶处理、纯化后测定浓度,RT-PCR验证。结果获得含H5N1禽流感病毒HA、NA、M全长基因序列的RNA片段,并可准确定量其拷贝数,质量浓度HA为503.9 ng/μL、NA为379.2 ng/μL、M为437.8 ng/μL。结论获得的RNA片段可作为H5N1禽流感病毒核酸快速检测方法的阳性定量标准品。

Objective To transcript RNA in vitro in order to obtain quantitative standard for detecting mRNA of H5N1 avian influenza virus.Methods According to the specific sequence of HA/NA/M gene of H5N1 avian influenza virus,the primers were designed and synthesized.The fragments generated by RT-PCR were cloned into the pGEM-T Easy vector with T7 cloning protocol.The positive recombinant linearized plasmids were used to transcript RNA in vitro and the RNA was used as standard quantitative template of RT-PCR method.Results Specific framents were amplified from virus culture medium,which were confirmed by DNA cloning and sequencing.The full-length cRNA of HA/NA/M gene of H5N1 avian influenza virus with precise quantification copy number was obtained successfully.Conclusion The HA/NA/M cRNA obtained could be used as quantitative standard for rapid detection of nucleic acid of H5N1 avian influenza virus.