一种改良方法克隆新的成人腹泻轮状病毒J19株的大片段基因

An improved method to amplify and clone large genome segments of a novel adult diarrhea rotavirus strain J19

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 large genome segments of a novel adult diarrhea rotavirus strain J19. Methods Using a novel adult diarrhea rotavirus strain J19 （NADRV-J19） as an example, 7 small genes of NADRV-J19 were cloned and sequenced by using the single-primer sequence-independent strategy; seven pairs of inhibitory primers from gene 5 to gene 11 were used to prevent amplification of their corresponding genes and improve amplification of large genome segments in one-tube polymerase chain reaction （PCR）. Results When two pairs of inhibitory primers were used to inhibit amplification of two corresponding small genes, yields of PCR products for large genome segments more than 2 kb were significantly improved in one-tube PCR. PCR products of genes were doned into pMD18-T vector, and sequenced. Finally, 3 fill-length cDNA clones for gene 2, 3, and 4 of J19 were obtained. Conclusion The improved single primer sequence-independent method could be used to clone large genome segments for rotavirases.