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目的构建小鼠H2B-绿色荧光蛋白(GFP)真核表达载体,为动态观察小鼠细胞中染色体的形态变化,从而进一步研究小鼠耐药细胞中双微体(DMs)的形成机制提供有效的分子工具。方法利用RT-PCR的方法获得小鼠H2B cDNA,以羧基端插入pcDNA3.1/CT-GFP-TOPO载体上,构建H2B-GFP真核表达载体,经菌液PCR、酶切及DNA测序鉴定插入片段大小、方向及序列的正确性,提取质粒转染小鼠胚胎成纤维细胞系NIH3T3进行鉴定。结果经菌液PCR、酶切和测序,证明小鼠H2B-GFP真核表达载体含有大小、方向及序列正确的H2B cDNA片段,转染NIH3T3后在细胞核中表达。结论成功构建了同时携带有G418筛选位点及多酶切位点的小鼠H2B-GFP真核表达载体,为其在小鼠体外培养细胞中的表达奠定了基础。
Objective To construct eukaryotic expression vector of mouse H2B-green fluorescent protein (GFP) for the purpose of dynamically observing the morphological changes of chromosomes in mouse cells so as to further study the formation mechanism of double microdimers (DMs) in mouse multidrug resistant cells and provide an effective Molecular tools. Methods H2B cDNA was obtained by RT-PCR and inserted into the pcDNA3.1 / CT-GFP-TOPO vector to construct H2B-GFP eukaryotic expression vector. The recombinant plasmid was identified by PCR, restriction enzyme digestion and DNA sequencing Fragment size, orientation and sequence correctness, the plasmid was transfected into mouse embryonic fibroblast cell line NIH3T3 for identification. Results The bacterial H2B-GFP eukaryotic expression vector was confirmed to contain H2B cDNA fragment of the correct size, orientation and sequence by bacterial PCR, digestion and sequencing. The recombinant plasmid was transfected into NIH3T3 and expressed in the nucleus. Conclusion The mouse H2B-GFP eukaryotic expression vector carrying both G418 screening site and multiple restriction sites was successfully constructed, which laid the foundation for its expression in mouse in vitro culture cells.