摘要
目的克隆小鼠白细胞介素-18(mIL-18)基因,构建重组表达质粒,在结肠癌中表达重组小鼠白细胞介素-18(mIL-18),并对其生物学活性进行初步鉴定。方法利用RT-PCR方法从小鼠腹腔巨噬细胞(Mφ)扩增出成熟肽编码区cDNA(474 bp),克隆到表达载体pSecTag2B(含有分泌信号肽Igκ)中;然后转染成纤维细胞,培养上清用小鼠的IL-18 ELISA试剂盒检测分泌有活性的mIL-18。结果构建的pSecTag2B-mIL-18可在成纤维细胞中分泌表达,mIL-18可诱导T淋巴细胞分泌IFN-γ。结论成功构建pSecTag2B-mIL-18表达载体,表达的IL-18刺激T淋巴细胞分泌IFN-γ。
Objective To clone mouse IL-18 eDNA, construct recombinant mouse IL-18 expression vector and perform the bioassav of recombinant mouse IL-18 expressed in fibroblast. Methods Mouse IL-18 cDNA fragment （474bp）was amplified from Mψfrom abdominal cavity of mice by RT-PCR and sequenced by ABI PRIMTM377XL DNA Sequener. The cloned mIL-18 cDNA was inserted pSecTag2Beukaryotic expression vector, which was subsequently transduced into a fibroblast cell line. The high secretion of bioactive mIL-18 was assayed with ELISA kit. The trandfected supernatant were added into T lymphocytes that were separated from mouse spleen. Results T lymphoeytes secreted lFN-Tin high level. Conlusion Successfully reconmbanited pSecTag2B-m-IL- 18 mammalian expression vectors, which secrets IL- 18 can stitmulate T lymphocytessecrete IFN-γ.
出处
《潍坊医学院学报》
2006年第6期418-420,I0002,共4页
Acta Academiae Medicinae Weifang
基金
山东省卫生厅基金资助项目(2001CA1DABB1)
