人类O^6-甲基鸟嘌呤-DNA-甲基转移酶蛋白的亚细胞荧光定位

Subcellular localization of human O^6-methylguanine-DNA-methyltransferase protein by fluorescent distribution

目的:机体对烷化剂诱导基因突变的修复能力取决于细胞内O6-甲基鸟嘌呤-DNA-甲基转移酶的含量及合成速度。构建人类O6-甲基鸟嘌呤-DNA-甲基转移酶基因与增强型绿色荧光蛋白基因融合表达的载体,根据荧光分布确定其亚细胞定位。方法:实验于2006-12/2007-06在南阳医学高等专科学校生理学实验研究中心完成。参考人类O6-甲基鸟嘌呤-DNA-甲基转移酶的基因序列和p-EGFP-N1载体的多克隆位点设计PCR扩增引物。从Hela细胞中提取总RNA,反转录合成O6-甲基鸟嘌呤-DNA-甲基转移酶的cDNA,进而合成双链DNA。扩增后的DNA目的片段与p-EGFP-N1载体均行NheI和BamHI双酶切,回收,定量后用T4连接酶连接,产物转化大肠杆菌感受态细胞TOP10,挑选阳性克隆并提取质粒测序,构建成功的质粒绿色荧光蛋白位于O6-甲基鸟嘌呤-DNA-甲基转移酶蛋白的C末端。将pEGFP-N1-MGMT载体分别转染HEK293和Hela细胞,荧光显微镜下与转染空载体pEGFP-N1的细胞进行对比观察,荧光信号表达最高点时进行固定并对细胞核进行复染,观察亚细胞定位情况,同时免疫印迹分析蛋白的表达。结果:①RT-PCR检测:扩增的O6-甲基鸟嘌呤-DNA-甲基转移酶基因cDNA片段约为650bp,与预期相符。②克隆及序列鉴定:扩增的DNA片段与载体pEGFP-N1经过NheI和BamHI双酶切后,可连接得到重组的表达载体,命名为pEGFP-N1-MGMT。重组质粒的酶切产物有650bp,4700bp两条带,克隆结果正确。该质粒经测序确认正确。③免疫印迹分析:转染pEGFP-N1质粒的细胞表达增强型绿色荧光蛋白,相对分子质量约为27000;转染pEGFP-N1-MGMT融合蛋白质粒的细胞相对分子质量约为49000,证明目的蛋白在转染细胞内表达成融合蛋白。④亚细胞定位:转染pEGFP-N1质粒的细胞绿色荧光信号均匀分布于整个细胞。转染pEGFP-N1-MGMT质粒的细胞绿色荧光主要集中在胞质,特别在一定条件下核周围有颗粒样聚集。结论:①实验成功构建pEGFP-N1-MGMT融合蛋白质粒。②人类O6-甲基鸟嘌呤-DNA-甲基转移酶基因定位在胞浆,且在一定条件下可以在核周聚集。

AIM： The ability of reparation of gene mutation induced by alkylation reagents lies on the quantity and synthetic rate of Oe-methylguanine-DNA-methyltransferase （MGMT） protein in organism cells. This study was designed to construct the expressed vector of the MGMT gene and enhanced green fluorescent protein （EGFP） gene fusion, and investigate the sub-cellular localization of human MGMT protein by fluorescent distribution. METHODS： The experiment was carried through from December 2006 to June 2007 at Experiment and Research Center of Physiology, Nanyang Medical College, Henan Province. According to the encoding sequence of the human MGMT gene and the multiple cloning sites of p-EGFP-N1 vectors, the two primers of the RT-PCR was designed. The total human RNA were extracted from Hela cells, and then amplified. MGMT RT-PCR product was re-ligated into Nhel and BamHI digested pEGFP-N1-vector by T4-1igase. The recombined plasmid was picked up and verified using the DNA sequencing analysis method. The EGFP gene was after the MGMT cDNA. HEK293 and Hela cells were transfected with pEGFP-N1-MGMT and pEGFP-N1 vectors and detected by fluorescence microscope when the signal of fluorescence was much stronger, fixed and dyed the cells, respectively. Meanwhile, sub-cellular localization was determined, and Westem-blotting results would make sure that the MGMT fused protein was expressed successfully. RESULTS： ①RT-PCR： The products of human MGMT cDNA were 650 bp according to the standard sequence. ②Clone and DNA sequencing analysis： MGMT gene was PCR amplified from the RT-PCR product and re-ligated into a Nhel and BamHI digested pEGFP-N1-vector. The products of pEGFP-N1-MGMT plasmid digested by Nhel and BamHI were 650 bp and 4 700 bp. The results were correct. Constructed integrity was verified using the DNA sequencing analysis method. ③Westem BloWing： The blot of EGFP protein, which molecular weight was 27 kD, was detected; while the blot of MGMT-EGFP fused protein was seen at the place of 49 kD. It was believed that the fused protein was expression in the HEK293 cells successfully. ④Sub-cellular localization： EGFP protein was distributed uniformly in the whole cells, while the MGMT-EGFP protein located in cytoplasm and there were some granules around the nucleus under some specific conditions. CONCLUSION： ①The plasmid of MGMT cDNA fused EGFP encoding sequence had been successfully cloned. ②The MGMT protein was distributed mainly in cytoplasm and there were some granules around the nucleus under some conditions.