重组人KNDC1腺病毒表达载体的构建及其促HUVEC衰老作用

Construction of human recombinant KNDC1adenovirus and its effect of promoting HUVEC senescence

目的构建人KNDC1腺病毒表达载体,观察其在人脐静脉内皮细胞(HUVEC)中的表达及功能。方法扩增KNDC1基因并与腺病毒骨架载体进行体外重组连接,经筛选、测序确认后转染进HEK293A细胞进行包装;按p Adeno X试剂盒的步骤纯化重组腺病毒,通过终点稀释法测定病毒滴度;用p AdenoⅩ-KNDC1感染HUVEC,48 h后用Western blot检测KNDC1蛋白表达水平;用SA-β-gal染色检测细胞衰老情况。结果经测序确认目的基因KNDC1成功克隆入腺病毒载体中。转染HEK293A细胞后观察到细胞变圆、部分细胞漂浮的特征性病变效应,病毒滴度达到1×1011PFU。感染了p AdenoⅩ-KNDC1的HUVEC中KNDC1蛋白表达水平显著升高(P<0.05)。随着p AdenoⅩ-KNDC1剂量的增加,衰老HUVEC数量增多。结论人KNDC1腺病毒表达载体构建成功并能促进HUVEC衰老。

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 HEK293 A cells for packaging. PAdeno Ⅹ-KNDC1 was purified by p Adeno Ⅹ kits and the endpoint dilution method was used for titering recombinant p Adeno Ⅹ-KNDC1. For observing the expression level of KNDC1 proteins,p Adeno Ⅹ-KNDC1 was used to infect HUVECs and then examined by Western blot. The function of p Adeno Ⅹ-KNDC1 was detected by senescence β-galactosidase staining kit. Results Gene sequencing indicated that p Adeno Ⅹ-KNDC1 contained KNDC1 gene. HEK293 A cells turned round 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 48 h after infection was observed. The number of senescence cells was increased with the increase of p Adeno Ⅹ-KNDC1 infection.Conclusions PAdeno Ⅹ-KNDC1 is successfully constructed and can promote the senescence of HUVECs.