携带人白细胞介素-1受体拮抗剂基因腺病毒质粒的构建

目的构建携带人白细胞介素-1受体拮抗剂(hIL-1Ra)基因重组腺病毒质粒。方法用EcoRⅤ和HindⅢ双酶切质粒pcDNA3-hIL-1Ra,获得hIL-1RacDNA片段,并定向连接在pShuttle-CMV穿梭载体上,构建穿梭质粒pShuttle-CMV/hIL-IRa。经PmeⅠ酶切线性化,穿梭质粒pShuttle-CMV/hIL-1Ra与超螺旋腺病毒骨架质粒pAdEasy-1共转化大肠杆菌BJ5183,二质粒在细菌内同源重组,得到重组pAd/hIL-1Ra腺病毒质粒。脂质体介导pAd/hIL-1Ra质粒转染HEK293细胞,包装产生复制缺陷型重组腺病毒,经反复感染HEK293细胞扩增病毒后,氯化铯密度梯度离心法纯化病毒,并测定病毒颗粒数、纯度及滴度。结果经PCR鉴定、限制性酶切分析及序列测定,证明已正确构建重组pShuttle-CMV/hIL-1Ra穿梭质粒和重组pAd/hIL-1Ra腺病毒质粒。扩增纯化后,重组腺病毒颗粒数为1.19×1011OPU/ml,A260/A280为1.279,滴度为3.6×109CCID50/ml。结论已成功构建重组腺病毒质粒pAd/hIL-1Ra,为hIL-1Ra基因在腺病毒载体介导下的免疫抑制治疗研究奠定实验基础。

Construction of Recombinant Adenovirus Vector Carrying Human Interleukin-1 Receptor Antagonist Gene

Objective To construct the recombinant adenovirus vector carrying human interleukin-1 receptor antagonist(hIL-1Ra)Methods Digest recombinant plasmid pcDNA3-hIL-1Ra with EcoR V and HindⅢ,and insert the obtained hIL-1Ra cDNA fragment into shuttle vector pShuttle-CMV.Co-transform E.coli BJ5183 with the constructed shuttle plasmid pShuttle-CMV/hIL-1Ra after linearization with Pme I and supercoil adenovirus skeleton plasmid pAdEasy-1 for homologous recombination.Transfect HEK293 cells with the obtained recombinant adenovirus vector pAd/hIL-1Ra in mediation of liposme.Propagate the produced replication-defective recombinant adenovirus by repeat infection of HEK293 cells and purify by cesium chloride gradient centrifugation,then count the vinus particles and determine the purity and titer.Results Both recombinant shuttle plasmid pShuttle-CMV/hIL-IRa and recombinant adenovirus vector pAd/hIL-IRa were correctly constructed as proved by PCR,restriction analysis and sequencing.After propagation and purification,the virus particle count,A260/A280 and titer of recombinant adenovirus were 1.19×1011 OPU/ml,1.279 and 3.6×109 CCID50/ml respectively.Conclusion Recombinant adenovirus vector pAd/hIL-IRa was successfully constructed,which laid a foundation of further study on adenovirus vector-mediated immunosuppressive therapy with hIL-IRa gene.