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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RAW 264.7中的表达。方法:从C57BL/6J小鼠胸腺组织提取总RNA,以RT-PCR法调取Ipr1基因编码序列,克隆至pEGFP-C1载体中,筛选阳性克隆作PCR、酶切及测序鉴定后得到pEGFP-Ipr1,脂质体瞬时转染小鼠巨噬细胞株RAW 264.7,以空质粒pEGFP-C1转染组作为对照。采用RT-PCR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-Ipr1,脂质体瞬时转染小鼠巨噬细胞株RAW 264.7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RAW 264.7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。
OBJECTIVE: To construct the fusion gene of Ipr1 gene and EGFP gene in mouse and to observe the expression of mouse Ipr1 gene in RAW 264.7 mouse macrophage cell line. Methods: The total RNA was extracted from the thymus of C57BL / 6J mice. The coding sequence of Ipr1 gene was obtained by RT-PCR and cloned into pEGFP-C1 vector. The positive clones were screened for PCR. After digestion and sequencing, pEGFP-Ipr1 The mouse macrophage cell line RAW 264.7 was transiently transfected with liposome and the empty plasmid pEGFP-C1 transfected group was used as a control. The mRNA level of Ipr1 was detected by RT-PCR and the expression of the fusion protein and the intracellular localization were observed by laser confocal microscopy. Results: The coding sequence of mouse Ipr1 gene was amplified. The correct recombinant plasmid pEGFP-Ipr1 was confirmed by PCR, restriction endonuclease digestion and sequencing. The RAW 264.7 transient transfection mouse macrophage cell line was successfully expressed. The expression of Ipr1 Gene expression products located in the nucleus. CONCLUSION: Mouse Ipr1 gene was successfully retrieved and the mouse Ipr1 gene and EGFP gene fusion expression vector was constructed and successfully expressed in murine macrophage cell line RAW 264.7. The Ipr1-encoded protein is localized in the nucleus, suggesting that it is a regulatory protein.