小鼠白细胞介素10重组腺病毒载体构建及对树突状细胞的基因修饰

Construction of mouse interleukin-10 recombinant adenovirus vector and gene modification for dendritic cells

背景:对于抗原递呈细胞树突状细胞及其分泌的细胞因子白细胞介素10在气道高反应性和炎症中的作用国内外文献报道较少。目的:构建小鼠白细胞介素10腺病毒重组体Ad-mIL-10,获得小鼠白细胞介素10基因修饰的树突状细胞,以期为下一步基因治疗的动物实验打下基础。方法:通过人工合成获得小鼠白细胞介素10基因,根据白细胞介素10基因序列及腺病毒载体的多克隆位点,合成包括酶切位点的基因序列,连接到pMD18-T载体并测序鉴定。用基因工程的方法将小鼠白细胞介素10基因克隆到BD Adeno-XTM腺病毒载体,于人胚肾293细胞中包装、扩增病毒并测定白细胞介素10蛋白表达,转染到体外培养的小鼠骨髓来源的树突状细胞。结果与结论:成功构建了小鼠Ad-mIL-10重组腺病毒载体并高包装、扩增成功,测定高表达白细胞介素10蛋白,体外成功培养小鼠骨髓来源的小鼠树突状细胞并顺利转染Ad-mIL-10。提示用基因工程方法构建小鼠Ad-mIL-10重组腺病毒载体并转染小鼠骨髓来源的树突状细胞是可行的,可为进行相关基因治疗的可能性提供更充足的理论依据。

BACKGROUND：Few reports concern the effects of dendritic cells-a kind of antigen presenting cells,and interleukin-10（IL-10） on airway hyperreactivity or inflammation.OBJECTIVE：To construct mice IL-10 recombinant adenovirus vector Ad-mIL-10 to acquire the dendritic cells modified by mIL-10,which can provide a foundation for the further study.METHODS：Mouse IL-10（mIL-10） gene comprise of enzyme cutting spot was synthesized according to the mIL-10 gene sequence and multiclone spot of adenovirus vector,connected to pMD18-T vector and sequenced.MIL-10 was subcloned to BD Adeno-XTM vector,packed and augmented in HEK 293 cells,following determine the protein expression,and the vector was transfected to mice bone marrow-derived dendritic cells.RESULTS AND CONCLUSION：Recombinant adenovirus vector Ad-mIL-10 was successfully synthesized,packed and augmented,which could highly express protein IL-10.Bone marrow-derived dendritic cells were successfully cultured and transduced in vitro.It suggested that it is feasible to transfect mice dendritic cells by Ad-mIL-10 adenovirus vector.The study can provide more sufficient theoretic evidence for the possibility of correlative gene therapy.