H5N1禽流感病毒抗原基因的体外转录及RNA标准品构建

In vitro transcription of HA,NA and M gene of H5N1 avian influenza virus and preparation of RNA standards

目的通过基因克隆和体外转录,获得H5N1禽流感病毒血凝素(HA)、神经氨酸酶(NA)、基质蛋白M(M)基因的RNA片段,为病原学检测提供阳性定量标准品。方法设计H5N1禽流感病毒的HA、NA及M基因全长开放阅读框的克隆引物,提取H5N1禽流感病毒总RNA,用RT-PCR获得相应片段,分别连接至pGEM-Teasy质粒,转化宿主菌XL10-Gold,然后提取阳性克隆的重组质粒DNA,测序鉴定后用限制性核酸内切酶NdeI酶切线性化,用T7RNA聚合酶进行体外转录,产物用DNase酶处理、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。结果获得含H5N1禽流感病毒HA、NA、M基因全长开放阅读框序列的准确定量拷贝数的RNA片段,质量浓度分别为503.9、379.2、437.8ng/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 obtain hemagglutinin （HA）, neuraminidase （NA） and matrix （M） gene segments of HSN1 avian influenza virus with gene cloning and in vitro transcription and to provide positive quantitative standard for pathogen detection. Methods According to the specific sequence of HA/NA/M gene of H5N1 avian influenza virus, the primers were designed and synthesized. Total RNA was extracted from H5N1 avian influenza virus and reverse transcribed to cDNA using RT-PCR. The HA/NA/M cDNAs were hgated with pGEM-T easy vector and transformed to bacterium XL10-Gold. Recombinant plasmid DNA extracted from positive clones was sequenced and digested with endonucleases Nde I. The linearized plasmids were used to transcript RNA iu vitro by T7 RNA potymerases, then the products were purifieated and fragmented with DNase for the detection of the concentration of cRNAs. Standard curves were constructed using Real-time fluorescence quantitative RT-PCR. Results The full-length cRNAs of HA/NA/M genes of H5N1 avian influenza virus were obtained by in vitro transcription with precise quan- tification copy number. The mass concentrations of cRNAs of HA/NA/M genes were 503.9, 379.2 and 437.8 ng/μl. Conclusion HA/ NA/M cRNA prepared can be used as positive quantitative standard for the rapid detection of nucleic acid of H5N1 avian influenza vinls.