BALB/c小鼠sIL-4受体基因克隆及腺病毒载体的构建

目的：克隆小鼠sIL-4R基因并构建sIL-4R基因腺病毒穿梭质粒，将之包装至重组腺病毒颗粒。方法：从小鼠脾脏细胞中提取总RNA，逆转录为cDNA，应用巢式PCR的方法克隆sIL-4R基因。目的基因经限制性内切酶酶切，定向克隆至腺病毒穿梭载体。利用脂质体将含目的基因的穿梭质粒与腺病毒包装质粒pJM17共同转染腺病毒包装细胞293细胞，PCR方法鉴定重组腺病毒阳性克隆。结果：BALB／c小鼠的sIL-4RcDNA序列与文献报道的小鼠sIL-4RcDNA序列同源性达98％，有9个碱基不匹配，与小鼠IL-4R的同种异型体的膜外区100％同源。293细胞出现特征性细胞病变。PCR鉴定获得重组病毒颗粒。结论：获得了小鼠sIL-4R基因和含sIL-4R基因的腺病毒颗粒，为探讨应用腺病毒介导sIL-4R蛋白治疗过敏性疾病打下了实验基础。

Cloning of soluble interleukin 4 receptor gene and construction of adenovirus shuttle vector

Objective: To obtain soluble interleukin 4 receptor (sIL-4R) gene and to clone sIL-4R gene into adenovirus shuttle vector. Methods: sIL-4R cDNA fragment was isolated from spleen cells of BALB/c mouse by a series of DNA manipulation and co-transfection of plasmids into 293 cells with LipofectAmine. Recombinant sIL-4R adenovirus particles were generated by homologous recombination of pAd sIL-4R and pJM17 in 293 cells. Results: The sequence of recombinant sIL-4R shared 100% homology with the extracellular part of IL-4R allotype and shared 98% homology compared with the published data of IL-4R secreted from T cell line CTLL.Nine-base substitutions led to 3-amino acid variation in the sIL-4R protein sequence. 293 cells showed evidence of cytopathic effect after ten days of infection. Conclusion:sIL-4R gene was isolated and introduced into adenovirus genome. The recombinant adenovirus particles might be explored for its use in preventing atopic and infectious diseases.