汉坦病毒S基因的克隆及体外转录

Cloning and RNA Transcription in Vitro of S Gene of Hantavirus

目的:通过基因克隆和体外转录,获得汉坦病毒汉滩型76118株及汉城型R22株S基因的RNA全长cRNA,为汉坦病毒病原学检测提供阳性定量标准品。方法:设计汉滩型76118株和汉城型R22株S基因克隆引物,PCR获得相应片段,分别克隆至含双启动子的PCRⅡ载体中,测序鉴定无误后,重组质粒分别经内切酶SpeⅠ、SacⅠ线性化,用T7 RNA聚合酶进行体外转录,产物经DNase处理、纯化后测定浓度,经RT-PCR验证。结果:获得汉滩型76118株及汉城型R22株S基因的cRNA片段,并可准确定量其拷贝数,76118株和R22株的质量浓度分别为80、17.58 ng/μL。结论:获得的cRNA样品可作为汉坦病毒核酸快速检测方法的阳性定量标准品。

Objective： To obtain full length cRNA of S gene of Hantaan virus 76118 and Seoul virus R22 by gene cloning and transcripting RNA in vitro, and offer quantitative standards for detecting hantavirus. Methods： According to the specific sequence of S genome segments of hantavirus, the primers were designed and synthesized. The fragments generated by PCR and confirmed by DNA cloning and sequencing were cloned into the PCR Ⅱ vec tor with T7 cloning protocol The positive recombined plasmids linearized by incision enzyme Spe I and Sac Ⅰ, and the linearized plasmids were used to transcript RNA in vitro. The purified RNA were used as standards quan titative template of RT-PCR method. Results： The full length cRNA of S gene segment of hantavirus were ob tained successfully, with the precise mass concentration of 80 ng/μL of Hantaan virus 76118 and 17.58 ng/μL of Seoul virus R22. Conclusion： The S cRNA of hantavirus obtained could be used as quantitative standards for rap id detection of nucleic acid of hantavirus.