hTFPI-2基因重组腺病毒载体的构建和体外U937单核细胞的表达  

Construction of Replication-deficient Recombinant Adenovirus Vector with hTFPI-2 Gene by AdMax System and Expression in U937 Monocyt

作　　者：潘俊杰[1] 施海明[1] 罗心平[1] 马端[2] 梁旺[2] 张进[2] 朱军[1] 李剑[1] 

机构地区：[1]复旦大学附属华山医院心内科,上海200040 [2]复旦大学分子生物学教育部重点实验室,上海200032

出　　处：《生物医学工程学杂志》2011年第2期326-331,共6页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(30971249)

摘　　要：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。We tried to construct and identify the recombinant replication-deficient adenovirus vector coding for human tissue factor pathway inhibitor 2（hTFPI-2） gene by AdMax system in HEK293 cells.Firstly,we obtained hTFPI-2 gene from the recombinant plasmid pIRES2-EGFP-TFPI-2 by PCR using primers with restriction endonuclease site of EcoRI or SacI.After digesting the hTFPI-2 gene and plasmid PDC316-IRES-EGFP shuttle vector,we ligated them with T4 ligase and formed the recombinant shuttle vector PDC316-IRES-EGFP-hTFPI-2.It was confirmed that the ligation product was inserted the gene of hTFPI-2 correctly by sequencing.Then we took cotransfection of HEK293 cells with the recombinant shuttle vector and genomic plasmid pBHGloxΔE1,3Cre by liposome lipofectamine2000,and finished the package of recombinant adenovirus Ad-hTFPI-2.The results of the PCR test and restriction endonuclease digestion confirmed the successful construction of the recombinants Ad-hTFPI-2.Furthermore,we measured the titre of Ad-hTFPI-2 with the aid of green fluorescence protein expression after multiplication and purification.The titre was 0.931×1012 pfu/ml.Finally,we infected U937 monocytes by purified Ad-hTFPI-2,and determined the infection efficiency and the TFPI-2＇s level and activity.The efficiency of Ad-hTFPI-2 infection in U937 cells was 89.33%.After infected by Ad-hTFPI-2,the TFPI-2＇s level in supernatant increased about 7 fold.Also the TFPI-2 in supernatant had activities of inhibiting trypsin and plasmin.The recombinant adenovirus with the hTFPI-2 gene was constructed successfully.It will be helpful for the further investigation of its potentiality to be applied in anti-atherosclerosis.

关 键 词：腺病毒载体 人组织因子途径抑制物-2 绿色荧光蛋白 噬斑 U937单核细胞 

分 类 号：R541.4[医药卫生—心血管疾病] Q782[医药卫生—内科学]