中文摘要：

目的构建小鼠血清类黏蛋白1样蛋白3（ORMDL3）基因的表达质粒,并在小鼠胚胎成纤维细胞（NIH3T3细胞）中检测其过表达效果。方法根据小鼠ORMDL3蛋白的编码序列设计引物,以NIH3T3细胞cDNA为模板,PCR扩增ORMDL3的编码序列,亚克隆入真核表达载体pcDNA3.1（＋）限制性内切酶KpnⅠ和HindⅢ酶切位点之间,并测序鉴定。由脂质体介导将pcDNA3.1（＋）和pcDNA3.1-ORMDL3质粒分别瞬时转染NIH3T3细胞中,24h后收取细胞,抽取细胞全蛋白,Western blot检测ORMDL3蛋白的相对表达量。结果成功构建小鼠ORMDL3表达质粒pcDNA3.1-ORMDL3,该质粒瞬时转染至NIH3T3细胞内可有效增加ORMDL3蛋白表达2倍左右（P〈0.05）。结论成功构建小鼠ORMDL3表达质粒,为后续小鼠动物模型研究ORMDL3基因的功能及致病机制提供依据。

英文摘要：

Objective To construct mouse orosomucoid 1-like protein 3（ORMDL3）expression plasmid.Methods The coding sequence of mouse ORMDL3（CCDS25355.1）was amplified by RT-PCR on mRNA from mouse embryonic fibroblast cells（NIH3T3 cells）.The products were purified and digested with KpnⅠ and HindⅢ.The digested fragments were subcloned into the pcDNA3.1（＋）expression vector,and the nucleotide sequence of the expression plasmid pcDNA3.1-ORMDL3 was confirmed by sequencing.The expression plasmids pcDNA3.1-ORMDL3 and pcDNA3.1（＋）empty vector were transfected into NIH3T3 cells by Lipofectamine 2000 reagent.Twenty-four hours after transfection,protein was extracted and the overexpression effect of pcDNA3.1-ORMDL3 was examined by Western blot.The empty vector of pcDNA3.1（＋）was used as the negative control.Results Sequence analysis showed that ORMDL3 coding sequence was accurately inserted into pcDNA3.1（＋）plasmid.Western blot indicated that pcDNA3.1-ORMDL3 could effectively increase ORMDL3 about 2times（P〈0.05）.Conclusion Expression vector pcDNA3.1-ORMDL3 is successfully constructed,which provides a foundation for subsequent studying on the function and pathogenic mechanism of ORMDL3 gene in mouse model.