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采用AdMax系统以复制缺陷型腺病毒为载体构建含有人组织因子途径抑制物-2(hTFPI-2)基因的腺病毒载体。用含EcoRI、SacI酶切位点的引物从pIRES2-EGFP-TFPI-2质粒中PCR扩增目的基因hTFPI-2,然后以限制性内切酶EcoRI和SacI消化hTFPI-2基因片段及腺病毒穿梭质粒PDC316-IRES-EGFP,用T4连接酶连接形成重组穿梭质粒PDC316-IRES-EGFP-hTFPI-2,与腺病毒骨架质粒pBHGloxΔE1,3Cre用脂质体Lipofectamine2000共转染HEK293细胞,包装成重组体腺病毒Ad-hTFPI-2。测序结果证明hTFPI-2序列正确,PCR、HindⅢ和Pac I酶切电泳证实腺病毒重组质粒Ad-hTFPI-2构建成功。经过扩增和CsCl结合法纯化后,利用穿梭质粒PDC316-IRES-EGFP中带有绿色荧光蛋白检测表达,测定病毒滴度为0.931×1012pfu/ml。重组病毒Ad-hTFPI-2对U937单核细胞感染率为89.33%。感染重组病毒较未感染时的上清TFPI-2浓度上升7倍左右,且对胰蛋白酶和纤溶酶具有抑制活性。成功地构建了能高效在U937单核细胞中表达hTFPI-2基因的重组腺病毒载体,为TFPI-2抗动脉粥样硬化作用的研究奠定基础。
The AdMax system was used to construct the adenoviral vector containing human tissue factor pathway inhibitor-2 (hTFPI-2) gene by using replication-deficient adenovirus as a vector. The target gene hTFPI-2 was amplified by PCR from the pIRES2-EGFP-TFPI-2 plasmid with primers containing EcoRI and SacI restriction sites. The hTFPI-2 gene fragment was digested with restriction endonucleases EcoRI and SacI and the adenovirus shuttle Plasmid PDC316-IRES-EGFP was ligated with T4 ligase to form recombinant shuttle plasmid PDC316-IRES-EGFP-hTFPI-2, which was cotransfected into adenovirus backbone plasmid pBHGloxΔE1,3Cre and transfected into HEK293 cells with Lipofectamine 2000, Virus Ad-hTFPI-2. Sequencing results showed that the hTFPI-2 sequence was correct, PCR, HindIII and Pac I electrophoresis confirmed the construction of recombinant adenovirus plasmid Ad-hTFPI-2. After purification by PCR and CsCl binding, the expression of green fluorescent protein in shuttle plasmid PDC316-IRES-EGFP was detected, and the virus titer was 0.931 × 1012pfu / ml. Recombinant virus Ad-hTFPI-2 on U937 monocyte infection rate was 89.33%. The concentration of TFPI-2 in the supernatant of the infected recombinant virus was 7 times higher than that in the non-infected and had inhibitory activity on trypsin and plasmin. The successful construction of a recombinant adenovirus vector that efficiently expresses hTFPI-2 gene in U937 monocytes provides a basis for the study of anti-atherosclerotic effects of TFPI-2.