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目的构建含单核苷酸多态性(Single nucleotide polymorphism,SNP)位点不同碱基的37 kD的N-牙本质基质蛋白1(Dentin matrix protein 1,DMP1)和全长DMP1基因重组表达质粒,并分析重组蛋白在HEK293细胞中的表达及定位。方法利用定点突变改造DMP1基因,获得DMP1基因的SNP rs10019009的不同基因型,将含SNP位点不同碱基的37 kD N-DMP1和全长DMP1基因定向克隆入含增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)的质粒pcDNA3.1-EGFP中,构建重组质粒pcDNA3.1-DMP1-EGFP,通过脂质体法瞬时转染HEK293细胞,荧光显微镜观察重组融合蛋白DMP1-EGFP的表达及胞内定位。结果 DNA测序结果表明,定点突变后的DMP1基因的碱基序列与设计序列完全一致;PCR和DNA测序显示重组质粒pcDNA3.1-DMP1-EGFP构建正确;融合蛋白DMP1-EGFP在HEK293细胞中主要表达于细胞胞浆中。结论成功构建了含rs10019009-SNP位点不同碱基的37 kD的N-DMP1和全长DMP1基因重组表达质粒,并在HEK293细胞的胞浆中有效表达,为进一步研究DMP1基因的功能奠定了基础。
Objective To construct a 37 kD Dentin matrix protein 1 (DMP1) and a full-length DMP1 gene recombinant plasmid containing different bases of single nucleotide polymorphism (SNP) The expression and localization of recombinant protein in HEK293 cells were analyzed. Methods The DMP1 gene was modified by site-directed mutagenesis to obtain different genotypes of SNP rs10019009 in DMP1 gene. The 37 kD N-DMP1 and the full-length DMP1 gene with different SNP sites were cloned into enhanced green fluorescent protein (EGFP) plasmid pcDNA3.1-EGFP. The recombinant plasmid pcDNA3.1-DMP1-EGFP was constructed and transfected into HEK293 cells by lipofectamine. The expression of recombinant fusion protein DMP1-EGFP and intracellular Positioning. Results The DNA sequencing results showed that the nucleotide sequence of DMP1 gene after site-directed mutagenesis was identical with the designed sequence. The PCR and DNA sequencing showed that the recombinant plasmid pcDNA3.1-DMP1-EGFP was constructed correctly. The fusion protein DMP1-EGFP was mainly expressed in HEK293 cells In the cytoplasm of cells. Conclusion The 37 kD N-DMP1 and full-length DMP1 recombinant plasmids containing different bases of rs10019009-SNP were successfully constructed and efficiently expressed in the cytoplasm of HEK293 cells, which laid the foundation for further study on the function of DMP1 gene .