论文部分内容阅读
目的构建一种含H7N9禽流感病毒全长血凝素/神经氨酸酶/基质蛋白(HA/NA/M)基因的重组质粒,为核酸检测方法提供一种通用的阳性定量标准品。方法设计H7N9禽流感病毒HA、NA及M抗原基因全长开放阅读框的克隆引物,提取H7N9禽流感病毒总RNA后用实时荧光定量PCR获得相应片段,用3次酶切连接方法,依次插入到pGEM-T easy质粒,进行测序确认;线性化后的重组质粒用T7 RNA聚合酶进行体外转录,RNA转录产物纯化后测定浓度,用实时荧光定量PCR构建标准曲线进行验证。结果 HA、NA和M基因扩增片段大小分别约为1.7、1.3、1.1 kb,与预期相符;构建的重组质粒pGEM-HA-NA-M插入片段的测序结果与GenBank公布序列一致;由重组质粒体外转录获得同时含有H7N9禽流感病毒HA、NA、M全长开放阅读框序列的RNA片段质量浓度为399.5 ng/μL,梯度稀释后用3种实时荧光定量PCR方法均获得了良好的标准曲线。结论成功构建重组质粒pGEM-HA-NA-M,由此质粒体外转录获得的RNA片段可作为H7N9禽流感病毒核酸快速检测方法通用的阳性定量标准品。
Objective To construct a recombinant plasmid containing the full-length hemagglutinin / neuraminidase / matrix protein (HA / NA / M) gene of H7N9 avian influenza virus and provide a universal positive quantitative standard for nucleic acid detection. Methods Clone primers of H7N9 avian influenza virus HA, NA and M antigen genes full-length open reading frame were designed. The total RNA of H7N9 strain was extracted and the corresponding fragments were obtained by real-time fluorescence quantitative PCR. The fragments were inserted into pGEM-T easy plasmid was confirmed by sequencing. The linearized recombinant plasmids were transcribed in vitro with T7 RNA polymerase. The RNA transcripts were purified and the concentrations were determined. The standard curve was constructed by real-time fluorescence quantitative PCR. Results The size of amplified fragments of HA, NA and M were about 1.7, 1.3 and 1.1 kb, respectively, in agreement with the expected results. The sequencing results of the constructed recombinant plasmid pGEM-HA-NA-M were consistent with those published in GenBank. In vitro transcription of the H7N9 avian influenza virus HA, NA, M full-length open reading frame RNA fragment mass concentration of 399.5 ng / μL, after gradient dilution using real-time fluorescence quantitative PCR 3 methods have obtained a good standard curve. Conclusion The recombinant plasmid pGEM-HA-NA-M was successfully constructed. The RNA fragment obtained from the plasmid in vitro can be used as a positive quantitative standard for the rapid detection of H7N9 avian influenza virus nucleic acid.