论文部分内容阅读
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 observe the effect of ZF-ATF, A20 gene, on the endogenous A20 gene and endothelial cell inflammatory response. Methods According to the sequence of human A20 gene, ZF-ATF was designed. Oligo DNA was synthesized and annealed to form double-stranded DNA. ZF-ATF eukaryotic vector was ligated with the vector which was digested with BamHⅠand KpnⅠ. Bacteria DH5α, screening positive clones, plasmid extraction, restriction endonuclease identification, identification of positive clones for sequencing. The plasmid DNA was transfected into human umbilical veinendothelial cells (HUVEC). The expression of A20 protein was detected by Western blot. The inflammatory response was induced by lipopolysaccharide (LPS) in HUVECs. The expression of NF-κB p65 was detected by Western blot The levels of TNF-α, IL-6 and IL-8 were detected by ELISA. Results Restriction endonuclease digestion and DNA sequencing confirmed that the synthetic ATF-containing eukaryotic vector oligonucleotide inserts were correctly inserted. Western blot confirmed that A20 protein expression was significantly increased after HUVEC transfection. The results of ELISA showed that the expression of IL-8, IL-6 and TNF-α in empty vector group and blank group were significantly increased after LPS stimulation, but not in ZF-ATF transfected group (P <0.05, P <0.01). Western blot showed that the expression of NF-κB p65 in ZF-ATF transfected group (19.8 ± 4.5) was significantly lower than that in empty vector group (90.2 ± 5.9) and blank group (96.3 ± 3.3) (P <0.01). Conclusion The eukaryotic expression vector of artificial zinc finger transcription factor was successfully constructed and could activate the stable expression of endogenous A20 and exert its anti-inflammatory effects.