关键词: KNDC1, 腺病毒, 同源重组, 脐静脉内皮细胞

Abstract: Objective To construct the human recombinant KNDC1 adenovirus and to investigate the expression and function of KNDC1 protein in human umbilical vein endothelial cells (HUVECs). Methods Human KNDC1 gene was firstly amplified by PCR and directly cloned into the linearized adenoviral vector in vitro, followed by screening and gene sequencing. Finally, the linearized recombinant plasmid was transfected into HEK293A cells for packaging. PAdenoⅩ-KNDC1 was purified by pAdenoⅩ kits and the endpoint dilution method was used for titering recombinant pAdenoⅩ-KNDC1. For observing the expression level of KNDC1 proteins, pAdenoⅩ-KNDC1 was used to infect HUVECs and then examined by Western Blot. The function of pAdenoⅩ-KNDC1 was detected by senescence β-galactosidase staining kit. Results Gene sequencing indicated that pAdenoⅩ-KNDC1 contained KNDC1 gene. HEK293A cells turned ground and partially floated in the tenth day after transfection. The titer was 1×1011 PFU. Meantime, significant upregulation of KNDC1 protein expression in HUVECs at 48h after infection was observed. The number of senescence cells was increased with the increase of pAdenoⅩ-KNDC1 infection. Conclusion PAdenoⅩ-KNDC1 was successfully constructed and could promote the senescence of HUVECs.

Key words: KNDC1, Adenoviruses, Homologous recombination, Human umbilical vein endothelial cells