携带小鼠白细胞介素-12 siRNA基因重组腺病毒载体的构建及其在树突状细胞中的表达

目的:构建载有白细胞介素-12干扰RNA(IL-12si RNA)基因的,能在树突状细胞(DC)表达的重组腺病毒载体,为免疫耐受提供基础。方法:将IL-12p35si RNA和IL-12p40si RNAcDNA克隆于腺病毒穿梭质粒pShuttle2,得到重组质粒,并与腺病毒骨架质粒BD Adeno-XTM体外连接后代共转化大肠杆菌DH5α菌株,重组腺病毒质粒经过293细胞的包装扩增及氯化铯(CsCl)纯化,生成带有IL-12p35和IL-12p40si RNA基因的重组腺病毒载体,感染DC细胞。结果:经形态学、病毒DNA酶切、聚合酶链反应(PCR)和逆转录(RT)等方法的鉴定,证实了载体构建的正确性;经测定,病毒滴度为2.5×1010efu/ml。结论:成功构建带有IL-12p35si RNA和IL-12p40si RNAcDNA的重组腺病毒载体,其所携带的载体能够转染小鼠DC并干扰其在DC中表达,为进一步的研究奠定了基础。

Construction of Adenoviral Vector Carrying Murine IL-12 SiRNA and Its Expression in Dendritic Cells

Objective: To develop an adenoviral vector for gene transfer of IL-12 siRNA and transfection to murine dendritic cells.Method: Murine IL-12p35 siRNA cDNA and IL-12p40 siRNA cDNA were inserted into adenovirus shuttle plasmid pShuttle2 to generate a recombinant plasmid pShuttle2 expression cassette, which is excised from pShuttle2 and ligated to BD Adeno-X Viral DNA by T4 Ligase , transformed chemically to DH5α cells .In The final stage, The recombinant BD Adeno-X vector is packaged into infectious adenovirus by transfecting human embryonic kidney (HEK) 293 cells and purificated by CsCl density gradient centrifugation. The expression of IL-12p35 siRNA and IL-12p40 siRNA in infected murine dendritic cells was detected by RT-PCR.Results: The morphology ,restriction analysis and RT-PCR were verified the successful construction and the titer of adenoviral was 2.5×10~ 10 efuml,The IL-12p35 mRNA and IL-12p40 mRNA were inhibited in the infected murine dendritic cells.Conclusion: The recombinant adenoviral vectors carrying IL-12p35 siRNA and IL-12p40 siRNA can be constructed successfully and transfected into murine dendritic cells.