可诱导表达mIL-12单链融合蛋白的腺病毒载体的构建

目的：构建带mIL-12单链融合蛋白基因并可经RU486诱导表达的重组腺病毒载体,以期用于体内的基因治疗研究。方法：利用分子生物学技术，构建了穿梭质粒PDC-RUmIL-12，使mIL-12单链融合蛋白基因和启动子在RU486诱导表达系统调控之下，酶切、测序鉴定正确后，将其与Admax腺病毒包装系统的辅助质粒共转染293细胞后，构建出可调控的重组腺病毒载体。用mIL-12引物对病毒进行了鉴定,扩增后用氯化铯密度梯度离心纯化病毒，采用最终稀释法测定了其滴度，并在结肠癌SW620细胞中用westen blot和ELISA方法检测了其基因调控效果。结果：经PCR鉴定证实可调控腺病毒载体Ad-RUmIL-12构建正确，病毒滴度达到4.62×1010pfu/ml。当给予诱导剂RU486后，腺病毒载体可以诱导表达mIL-12单链融合蛋白，表达量随着诱导剂浓度而增加，而没有RU486时，几乎没有mIL-12蛋白的表达。结论:成功构建了重组腺病毒 Ad-RUmIL-12，可以通过RU486诱导mIL-12单链融合蛋白的表达，为下一步体内基因治疗奠定了良好的基础。

Construction a RU486 inducible recombinant adenovirus of single chain fusion murine IL-12 protein

Objective To construct a RU486 inducible recombinant adenovirus of single single chain fusion murine IL-12 protein for future gene therapy in vivo. Methods : The shutter vector PDC-RUmIL-12 was constructed by molecular biology technique. The single chain murine IL-12 and hCMV promoter were controlled under the RU486 inducible system. The positive clone of the shutter vector was identified by restriction endonuclease digestion and further confirmed by sequencing. Recombinant adenovirus were produced after cotransfection of the shutter vector PDC-RUmIL-12 and adenovirus DNA helper plasmid pBHGloxΔE1,3Cre into 293 cells .The recombined adenovirus was purified by CsCl density gradient centrifugation and its titer was determined by end point dilution assay. Expression of this regulatable recombinant adenovirus vector in infected SW620 cells was tested by the ELISA kit and westen blot. Results: The adenovirus vector containing single single chain fusion murine IL-12 gene was identify by PCR. The virus titer of Ad-RUmIL-12 was 4.62×1010 pfu/ml. The quantity of IL-12 protein expression was increased with the concentration of inducer, whereas no significant IL-12 protein was detected without the absence of RU486. Conclusion :A RU486 regulatable replication-defective recombinant adenoviruses vector containing single chain fusion murine IL-12 gene was successfully constructed，which provides a good basis for further research on gene therapy in vivo.