应用细菌内同源重组机制快速克隆腺病毒基因组的研究

A Rapid Method for Cloning the Adenoviral Genomes by Bacterial Intermolecular Homologous Recombination

A novel procedure was used for cloning large adenovirus genome fragment by the homologous recombination in E.coli strain BJ5183. The 11.2Kb downstream fragment of the CAV-2 strain YCA18 genome was cloned by homologous recombination, the 1029bp left end and the 970bp right end of this fragment were separately amplified by PCR. They were then cloned into plasmid pPoly2 with direction from left fragment to right fragment, obtaining a“rescue”plasmid pT615. The pT615 was liberalized by Hind Ⅲ and PstI digestion and was cotransformed with the purified CAV-2 genome which was cut by BstBI into competent E.coli strain BJ5183. Recombinant plasmids harboring the 11.2Kb downstream fragment of CAV-2 genome were obtained after bacterial intermolecular homologous recombination. The recombinant efficiency of all E.coli strains tested was 78.3%. One of the recombinant plasmids, pT618, was further identified by enzyme digestion analysis and PCR amplification. The results showed the plasmids contained the 11.2kb fragment downstream the genome of CAV-2.