可诱导表达mIL-12单链融合蛋白的腺病毒载体的构建

Construction of a recombinant adenovirus with RU486 inducible expression of single chain fusion murine interleukin-12 protein

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:构建穿梭质粒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HEK293细胞后,构建出可调控的重组腺病毒载体,用mIL-12引物对病毒进行鉴定。氯化铯密度梯度离心纯化病毒,最终稀释法测定其滴度,并在结肠癌SW620细胞中Western Blot和ELISA法检测其基因调控效果。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。

Objective To construct a recombinant adenovirus carrying the gene of single chain fusion murine interleukin-12（IL-12） protein, which can be expressed through the induction of RU486, for using in future gene therapy in vivo. Methods The shutter vector pDC-RUmIL-12 was constructed by molecular biology technique. The gene of single chain fusion murine IL-12 protein 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 pBHGlox△E1 ,E3cre into HEK293 cells. The recombined adenovirus was purified by CsC1 density gradient centrifugation and its titer was determined by end-point dilution assay. Expression of this regulable recombinant adenovirus vector in infected SW620 cells was tested by the ELISA kit and western blot. Results The adenovirus vector containing single chain fusion murine IL-12 gene was identified by PCR. The virus titer of Ad-RUmIL-12 was 4.62 ×10^10 pfu/mL. The quantity of IL-12 protein expression was increased with the increasing concentration of inducer, whereas no significant IL-12 protein was detected in the absence of RU486. Conclusion A RU486 regulable replication-defective recombinant adenoviruses vector containing single chain fusion murine IL-12 gene has been successfully constructed ,which provides a good basis for further research on gene therapy in vivo.