【摘要】 为了获取Ipr1基因全长编码序列,构建与EGFP基因融合的表达载体,观察小鼠Ipr1基因在小鼠巨噬细胞中的表达情况及对细胞生长状态的影响,试验通过RT-PCR方法获得Ipr1基因,将获得的Iprl基因片段与pEGFP-C1载体片段连接后获得真核表达质粒pEGFP-Ipr1,经PCR、酶切鉴定正确后,脂质体瞬时转染pEGFP-Ipr1至第5代的小鼠腹腔巨噬细胞内,以空质粒pEGFP-C1转染组作为对照,采用Western-blot检测Ipr1基因的表达并用荧光显微镜观察融合蛋白的表达情况。结果表明:研究构建了真核表达载体pEGFP-Ipr1并进行了Ipr1基因转染,通过Western-blot检测在大约50 ku处有目的条带,转染24 h后有绿色荧光蛋白表达,并经G418筛选获得稳定转染Ipr1基因的小鼠腹腔巨噬细胞株。说明试验获得了易于转染的小鼠腹腔巨噬细胞株,成功转染了Ipr1基因并得以表达。更多还原

【Abstract】 To obtain full length coding sequence of intracellular pathogen resistance 1( Ipr1) gene and construct the prokaryotic expression vector carrying Ipr1 and enhanced green fluorescent protein( EGFP) fusion gene,and observe the expression levels of Ipr1 gene in murine peritoneal macrophages and its effect on the growth state of the cells. Reverse transcription polymerase chain reaction( RT-PCR) method was used to obtained the Ipr1 gene,the obtained gene fragment from the Ipr1 gene was used to ligate the vector fragment of pEGFP-C1 to obtain eukaryotic expression plasmid pEGFP-Ipr1,and then was identified by PCR and restriction enzyme digestion. The pEGFP-Ipr1 was transiently transfected into peritoneal macrophages of the fifth generation of mice by liposomes. The pEGFP-C1 transfection group with empty plasmid was used as a control group,and then the expressions of Ipr1 gene and fusion protein were detected and observed using Western-blot and fluorescence microscopy,respectively. The results showed that the eukaryotic expression plasmid pEGFP-Ipr1 was constructed,and the Ipr1 gene was transfected. There was a target band at about 50 ku,and the expression of green fluorescent protein was found at 24 h after transfection. The peritoneal macrophage cell strain was obtained by G418 screening,which could stably transfect the Ipr1 gene. The results indicate that the peritoneal macrophage cell strain is successfully obtained,which is easy to transfect,and the Ipr1 gene is successfully transfected and expressed.更多还原