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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 2 93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 10 0 %同源。 2 93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。
OBJECTIVE: To clone mouse sIL_4R gene and construct sIL_4R adenovirus shuttle plasmid and package it into recombinant adenovirus particles. METHODS: Total RNA was extracted from mouse spleen cells and reverse transcribed into cDNA. The sIL_4R gene was cloned by nested PCR. The target gene was digested with restriction enzyme and cloned into the adenoviral shuttle vector. The shuttle plasmid containing the gene of interest was co-transfected with the adenoviral packaging plasmid pJM17 into 293 Adenovirus packaging cells using liposomes, and the recombinant adenovirus positive clones were identified by PCR. Results: The sequence of sIL_4RcDNA in BALB c mice was 98% homologous to the reported sIL_4RcDNA in mice, with 9 base pairs mismatched with 100% homology in the extramembranous region of mouse IL_4R allotype . 2 93 cells showed characteristic cytopathic effect, PCR identification obtained recombinant virus particles. CONCLUSIONS: Mouse sIL_4R gene and adenovirus particles containing sIL_4R gene were obtained, which laid the experimental foundation for exploring the application of adenovirus-mediated sIL_4R protein in the treatment of allergic diseases.