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目的 :建立扩增登革 2型病毒基因组全长cDNA的融合PCR法 ,为深入探讨病毒基因组的结构与功能提供有效的技术途径。方法 :根据我国登革 2型病毒D2 4 3株序列设计引物 ,首先采用长链RT PCR技术扩增病毒基因组 5′及 3′半分子 ,然后以半分子采用融合PCR法将两个半分子构建成全长cDNA分子。为进一步证实所构建全长cDNA分子的特异性 ,再以融合PCR产物为模板扩增 5′非编码区序列 ,与pGEM T载体连接 ,在 377A型自动测序仪进行序列分析。结果 :通过对逆转录反应及PCR扩增条件的优化 ,建立了制备大片段cDNA及构建全长cDNA分子的融合PCR方法。扩增的我国登革 2型病毒的 5′及 3′半分子的长度均为 5kb左右 ,采用融合PCR方法制备的全长cDNA长约 11kb ,与预期大小一致 ,非编码区测序结果表明扩增产物为D2 4 3所特有。结论 :所建立的融合PCR方法是构建登革病毒基因组全长cDNA的有效技术
OBJECTIVE: To establish a fusion PCR method for amplifying the full-length cDNA of dengue 2 virus genome and to provide an effective technical approach for further exploration of the structure and function of the virus genome. Methods: According to the sequence of dengue virus type 2 (D2 4 3) in China, primers were designed. First, the 5 ’and 3’ half of the viral genome was amplified by long-chain RT-PCR. Then two half molecules were constructed by fusion PCR Into full-length cDNA molecules. To further confirm the specificity of the constructed full-length cDNA molecules, the fusion PCR product was used as a template to amplify the 5 ’non-coding region sequence, ligated with the pGEM T vector and sequenced in a 377A automatic sequencer. Results: The fusion PCR method for preparation of large cDNA and full-length cDNA was established by optimizing the conditions of reverse transcription reaction and PCR amplification. The amplification of 5 ’and 3’ half of dengue virus type 2 in China is about 5kb in length. The full-length cDNA prepared by fusion PCR is about 11kb in length, consistent with the expected size. Sequencing results in non-coding region indicate that amplification The product is unique to D2 4 3. Conclusion: The established fusion PCR method is an effective technique to construct full-length cDNA of dengue virus genome