稳定表达细胞内病原体抗性基因1的RAW264.7细胞克隆的建立

Establishment of RAW264.7 cell clones stably expressing intracellular pathogen resistance 1

目的:构建携带细胞内病原体抗性基因1(Ipr1),以绿色荧光蛋白(GFP)为报告基因的重组真核表达载体,并导入鼠巨噬细胞RAW264.7中表达.方法:KpnⅠ和BamHⅠ双酶切重组质粒pET32a(+)-Ipr1及真核表达载体pEGFP-C1,Ipr1基因定向克隆入pEGFP-C1载体,构建重组质粒载体pEGFP-C1-Ipr1.脂质法转染体培养的RAW264.7细胞,在活细胞状态下用荧光显微镜直接观察pEGFP-C1-Ipr1融合蛋白在细胞中的表达;PCR检测Ipr1基因转录水平的表达;West-ern Blot方法验证Ipr1蛋白水平的表达.结果:酶切鉴定获得一条约4700bp空载体条带及一条约1338bp目的片段,证明pEGFP-C1-Ipr1真核表达载体构建成功.pEGFP-C1-Ipr1转染RAW264.7细胞后,Western Blot可以检测到约Mr77×103的融合蛋白在RAW264.7细胞中表达,荧光显微镜下可观察到转染细胞中有绿色荧光蛋白表达.结论:成功构建真核绿色荧光蛋白表达载体pEGFP-C1-Ipr1,获得稳定的RAW264.7-Ipr1细胞克隆可表达Ipr1,为进一步研究Ipr1的功能奠定了基础.

AIM ： To construct pEGFP-CI-Ipr1 （ intracellular pathogen resistance 1 ） eukaryotic expression vector using green fluorescence protein as reporter gene and to express it in RAW264.7 cells. METHODS： The pET32a （ ＋ ）-Iprl and pEGFP-C1 vectors were digested by Kpn I/BamH Ⅰ and purified by gel extraction. The Iprl genes were inserted into pEGFP-C1 vector. The RAW264.7 cells were transfected with the constrncts by Llpofectamine ^TM 2000. The EGFP-Iprl fused protein was visualized directly under fluorescence microscope and the expression of Iprl was detected by Western blot and PCR at protein and gene levels respectively. RESULTS： The fragments with size of 4700 bp and 1338 bp were obtained by enzyme digestion and a fusion protein about 77 ku was observed by Western Blot. Restriction enzyme digestion and sequence analysis showed that the recombinant vector pEGFP-CI-Iprl was constructed successfully, which was stably expressed in eukaryotic cells. Iprl gene and protein were detected in the transfected RAW264.7 cells and the expressed EGFP was observed under fluorescence microscope. CONCLUSION： The eukaryotic expression vector named pEGFP-CI-Iprl is successfully constructed and the RAW264.7 cell clone stably expressing Iprl is obtained, which provides a good basis for further research on the function of Iprl against tuberculosis.