Author(s): FENG Jie, ZHUANG Lili, ZHU Lianghua, Department of Pediatrics, First Affiliated Hospital, Nanjing Medical University
Pages: 125-128
Year: 2015 Issue:  2
Journal: Jiangsu Medical Journal
Keyword:  Orosomucoid 1-like protein 3; Asthma;
Abstract: 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.