利用搭桥PCR法实现轮状病毒VP7表位片段的重组

Multi-epitope Fragments of Rotavirus VP7 Recombination by over Lapextension PCR

选择了RV VP7的3个高度保守可诱发高水平体液免疫反应的中和性抗原表位,人工合成了这3个表位片段。采用了一种不需要限制性内切酶和连接酶的重组DNA方法—搭桥PCR法,方便快速地构建了轮状病毒VP7蛋白的重组表位基因,将其T/A克隆入PBS-T载体,经过测序与报道序列同源性达100%。结果表明:搭桥法PCR简单易行,快速准确,尤其是在构建2个或多个小基因片段的融合时,比酶切、连接方法更具优越性。

Three neutralizing epitopes of RV VP7 protein were chosen,which could induce effective humoral immunity. The epitopes from RV VP7 gene were synthesized and then linked them to a long epitope by over lapextension PCR. T/A cloning of the recombinant RV VP7 muti-epitopes gene have been finished successfully. The result showed that over lapextension PCR was easy and exact when constructed two or more short DNA fragments, which was superior to enzyme digestion and ligation.