Li Na, Zhu Dao-yin, Tie Ru-xiu, Wang Yu-wei
Department of Immunology, Chongqing University of Medical Sciences, Chongqing 400016, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Mar;24(3):231-3.
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.
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.
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.
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.
构建含小鼠Ipr1与增强绿色荧光蛋白（EGFP）融合基因的真核表达载体，并研究其在小鼠巨噬细胞RAW264.7中的表达情况。
通过逆转录聚合酶链反应（RT-PCR）从C57BL/6J小鼠胸腺总RNA中扩增Ipr1基因的编码序列。将该基因克隆至pEGFP-C1载体中，通过聚合酶链反应（PCR）、限制性内切酶消化及测序鉴定重组质粒。随后将pEGFP-Ipr1瞬时转染至RAW264.7细胞中。通过RT-PCR及激光扫描共聚焦显微镜检测Ipr1基因及融合蛋白的表达。
成功扩增出Ipr1的完整编码序列。通过PCR、限制性内切酶消化及测序鉴定重组质粒。融合蛋白在靶细胞中成功表达，且定位于细胞核。
成功构建了真核表达载体pEGFP-Ipr1。融合蛋白可在小鼠巨噬细胞RAW264.7中表达并定位于细胞核。
