A20人工锌指转录因子真核载体的构建及其对内皮细胞炎症反应的影响

Construction of recombinant plasmid ZF-ATF and evaluation of its role in anti-inflammation of HUVEC cell line

目的观察A20基因的人工锌指转录因子(zinc finger-artificial transcription factor,ZF-ATF)对内源性A20基因及内皮细胞炎症反应的影响。方法根据人A20基因的序列,设计出其ZF-ATF,合成靶序列Oligo DNA,退火形成双链DNA,与通过BamHⅠ和KpnⅠ酶切后的载体连接产生ZF-ATF真核载体,质粒转化感受态细菌DH5α,筛选阳性克隆,提取质粒,限制性内切酶酶切鉴定,鉴定阳性克隆进行测序。质粒DNA转染人脐静脉内皮细胞(human umbilical veinendothelial cell,HUVEC),通过Western blot检测A20蛋白的表达,用脂多糖(LPS)诱导HUVEC细胞产生炎症反应,利用Western blot检测NF-κB p65的表达情况,利用ELISA检测TNF-α、IL-6、IL-8等的水平。结果限制性内切酶酶切鉴定与DNA测序证实合成的含ATF的真核载体寡核苷酸链插入正确,Western blot证实转染HUVEC后A20蛋白表达显著增加。ELISA法检测结果表明,在LPS刺激后,空载体组及空白组细胞因子IL-8、IL-6、TNF-α表达均明显上调,而ZF-ATF转染组没有明显上调,与空载体组及空白组比较,有统计学差异(P<0.05,P<0.01);Western blot证实在LPS刺激后ZF-ATF转染组NF-κB p65的表达(19.8±4.5)较空载体组(90.2±5.9)及空白组(96.3±3.3)显著下调(P<0.01)。结论成功构建人工锌指转录因子真核表达载体,且能够启动内源性A20的稳定表达,并发挥其抗炎等作用。

Objective To construct an eukaryotic expression vector of A20 artificial zinc finger-artifi- cial transcription factor （ZF-ATF）, which expresses A20 stably, and evaluate its anti-inflammation effects on human umbilical vein endothelial cells（HUVEC）. Methods A20 artificial ZF-ATF was designed based on the gene sequence of A20. Then the sequence of ZF-ATF was subcloned into the eukaryotic expression vector PCDNA-NLS-P65-Flag, which was identified and confirmed by restriction enzyme digestion and DNA sequen- cing. Subsequently, the PCDNA-NLS-ZF-ATF-P65-Flag plasmid was transfected into HUVEC cells, and then the expression of protein A20 was detected by Western blotting. The expression of NF-KB p65 was detected in HUVEC cells after LPS treatment by Western blotting. The concentrations of TNF-ct, IL-6 and IL-8 in the supernatants were determined by cytokine ELISA kits. Results Restriction enzyme digestion and DNA sequencing confirmed that the obtained sequence of ZF-ATF was correct. Western blotting indicated that the expression of protein A20 was upregulated in HUVEC cells in 24- l~ after recombinant vector transfection. After stimulation with LPS for 24 h, the concentrations of TNF-c~, IL-6 and IL-8 in the supernatants of HUVEC cells were obviously increased in the blank and control groups, but those had no change in ZF-ATF group, which were significantly lower in the blank and control groups （P 〈 0, 05, P 〈 O. 0i ）. Western blotting indicated that LPS stimulation resulted in an sigfiifieant decrease of NF-KB p65 in ZF-ATF group （19.8± 4.5 ） than in blank （90.2 ±5.9） and contrQlgroups （96.3 ±3.3,, both P 〈0.01）. Conclusion The eukaryotic expression vector of ZF-ATF is sue.cessfully constructed, which expresses A20 stably and A20 could play crucial protective roles in anti-inflammation of HUVEC cells against LPS stimulation. This research could be a foundation for our study the molecular function of A20.