基于Red重组系统对HCMV BAC Towne UL49基因的改造

HCMV BAC Towne was committed to research in this topic by Red-recombination systems. The expression of genes mediating Red recombination was first induced by the addition of L-arabinose. Then,the linear rpsl-neo counter-selection cassette flanked by homologous arms "hm" was electroporated into the DH10B E.coli strain carrying HCMV BAC Towne and PKD46 plasmid. And Red recombination inserted the functional cassette into the target locus. Only colonies carrying the modified BAC will survive kanamycin selection on the agar plates and become streptomycin sensitive. The successful integration of the counter-selection cassette will be monitored by PCR and DNA sequencing. The correct colonies were named with BAC-Towne-DL49. We have successfully contructed a UL49-deletion mutant recombination. And the recombinant HCMV BAC Towne would be able to lay foundation for the research of HCMV UL49 gene function.