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登革病毒(Dengueviruses─DV)为单链正股RNA病毒,其非结构蛋白NS1在病毒免疫反应中起重要作用。我们在NS1基因序列设计了一对通用引物,扩增序列长度为413bP。应用逆转录──聚合酶链反应(RT─PCR)成功地扩增了DV1─4型部分基因片段,采用该引物扩增国内分离株DV1(GZ)和DV2(HN91)同样出现一条特异扩增带,回收该DNA片段,并将DV1(GZ)和DV2(HN91)NS1基因部分片段分别克隆到PUC19质粒载体中。经Pst1和EcoR1双酶切分析和通用引物PCR鉴定证明重组质粒内含有NS1基因部分片段。准备进行核甘酸序列分析。同时,以上无性繁殖系的构建,为DV的核酸杂交研究提供了特异性基因探针。
Dengue virus (DV) is a single-stranded single-stranded RNA virus whose nonstructural protein NS1 plays an important role in the viral immune response. We designed a pair of universal primers in the NS1 gene sequence, the amplification sequence length of 413bP. The DV1-4 partial gene fragment was successfully amplified by reverse transcription-polymerase chain reaction (RT-PCR). Using this primer to amplify the domestic isolates DV1 (GZ) and DV2 (HN91), a specific amplification The DNA fragment was recovered and the DV1 (GZ) and DV2 (HN91) NS1 gene partial fragments were respectively cloned into the PUC19 plasmid vector. The Pst1 and EcoR1 double digestion analysis and universal primer PCR identification showed that the recombinant plasmid contains NS1 gene partial fragment. Prepare for nucleotide sequence analysis. Meanwhile, the cloning of the above clones provided specific gene probes for the study of DV nucleic acid hybridization.