小鼠Ipr1基因与EGFP基因融合表达载体的构建及其在鼠巨噬细胞中的表达_临床医学论文.docVIP

小鼠Ipr1基因与EGFP基因融合表达载体的构建及其在鼠巨噬细胞中的表达_临床医学论文 小鼠Ipr1基因与EGFP基因融合表达载体的构建及其在鼠巨噬细胞中的表达_临床医学论文 作者：李娜, 朱道银, 帖儒修, 王瑜伟 【摘要】 　　目的: 构建小鼠胞内病原体抗性基因1（Ipr1）基因与EGFP基因融合表达载体, 观察小鼠Ipr1基因在小鼠巨噬细胞株RAW264.7中的表达。方法: 从C57BL/6J小鼠胸腺组织提取总RNA, 以RT法调取Ipr1基因编码序列, 克隆至pEGFP载体中, 筛选阳性克隆作PCR、 酶切及测序鉴定后得到pEGFP脂质体瞬时转染小鼠巨噬细胞株RAW264.7, 以空质粒pEGFP转染组作为对照。采用RT法检测Ipr1的RNA水平表达及用激光共聚焦显微镜观察融合蛋白的表达及细胞内定位。结果: 扩增出小鼠Ipr1基因编码序列, 经PCR、 酶切及测序鉴定证明得到正确的重组质粒pEGFP脂质体瞬时转染小鼠巨噬细胞株RAW264.7得到了成功表达, Ipr1基因表达产物定位于细胞核内。结论: 成功地调取小鼠Ipr1基因, 构建了小鼠Ipr1基因与EGFP基因融合表达载体并在小鼠巨噬细胞株RAW264.7得到了成功表达。Ipr1编码蛋白定位于细胞核内, 提示其是一种调节蛋白。 【关键词】 Ipr1基因 结核病 绿色荧光蛋白 定位 　　[Abstract]AIM: To construct an eukaryotic expression vector containing the fusion gene of mouse Ipr1 and EGFP and to study its expression in murine macrophage RAW264.7. METHODS: The coding sequence of Ipr1 gene was amplified from the total RNA of C57BL/6J mouse thymus by RTPCR. The gene was cloned into pEGFPC1 and the recombinant plasmid was identified by PCR, restrict endonuclease digestion and sequencing. Then the pEGFPIpr1 was transiently transfected into RAW264.7. The expression of Ipr1 gene and fusion protein was detected by RTPCR and laser scanning confocal microscopy. RESULTS: The whole coding sequence of Ipr1 was successfully amplified. The recombinant plasmid was identified by PCR, restrict endonuclease digestion and sequencing. The fusion protein was successfully expressed in the targeted cells and its localization was in nucleus. CONCLUSION: The eukaryotic expression vector pEGFPIpr1 has been successfully constructed. The fusion protein can be expressed in murine macrophage RAW264.7 and located in nucleus. 　　[Keywords]Ipr1 gene; tuberculosis; pEGFP 　　结核病位居单一病原体引起患者死亡的传染病之首, 全球每年死亡200余万人。我国属结核病高负担国家, 结核病感染人数居全球第二。感染结核分枝杆菌（Mycobacterium tuberculosis, Mtb）的人中仅10%的人发展为结核病,除环境等因素外, 遗传可能决定了机体的易感性[1]。2005年Pan等[2]在小鼠巨噬细胞内发现了一个介导巨噬细胞抗胞内病原体的基因, 称为胞内病原体抗性基因1（intracellular pathogen resistance 1, Ipr1）, 该基因与结核病的易感性有着密切的关系。Ipr1基因缺陷的小鼠感染Mtb后巨噬细胞表现为坏死, 从而有利于细菌在宿主体内的繁殖与扩散; 而表达Ipr1基因的小鼠, 巨噬细胞则发