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目的扩增、克隆新的成人腹泻轮状病毒J19株的大片段基因。方法以新成人腹泻轮状病毒J19株的病毒核酸为材料,利用非依赖核酸序列的单引物扩增方法,扩增新的成人腹泻轮状病毒J19株的第5-11基因;根据第5-11基因序列设计7对抑制性引物,通过在PCR扩增体系中添加小片段基因的抑制性引物来提高大片段基因的PCR产量。结果当在PCR反应体系中添加2对抑制性引物时,大于2kd的大片段基因的PCR产量得到显著提高。将扩增的基因片段克隆至pMD18- T后进行测序,最后得到J19株第2、3、4基因的全长eDNA克隆。结论这种改良的非依赖核酸序列的单引物扩增方法可以用于对轮状病毒大片段基因的扩增和克隆。
Objective To amplify and clone the large fragment gene of new adult diarrhea rotavirus strain J19. Methods The novel nucleic acid sequence of rotavirus strain J19 was used to amplify the 5-11 gene of the new adult diarrhea rotavirus strain J19 by using the single primer amplification method without any nucleic acid sequence. 11 Gene Sequence 7 pairs of inhibitory primers were designed to increase PCR yield of large fragment genes by adding inhibitory primers of small fragment genes to the PCR amplification system. Results When two pairs of inhibitory primers were added to the PCR reaction system, the PCR yield of large fragment genes larger than 2kd was significantly increased. The amplified gene fragment was cloned into pMD18-T for sequencing, and finally the full length cDNA clone of the 2,3,4 gene of strain J19 was obtained. Conclusion This improved non-nucleic acid sequence-based single primer amplification method can be used to amplify and clone the large fragment of rotavirus.