Construction of X IAP-3′UTR-luciferase reporter vector and its activity analysis
10.3969/j.issn.1673-4130.2014.09.004
- VernacularTitle:XIAP基因3′非翻译区荧光素酶报告载体的构建及活性分析
- Author:
Ning DONG
;
Gaoxia GE
;
Weiming ZHANG
;
Wei ZHU
;
Huaguo XU
- Publication Type:Journal Article
- Keywords:
X-linked inhibitor of apoptosis protein;
3′untranslational region;
microRNA,miRNAs;
luciferases,bacterial;
vectors
- From:
International Journal of Laboratory Medicine
2014;(9):1098-1099,1102
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant X-linked inhibitor of apoptosis protein(XIAP) gene 3′untranslational region (3′UTR)-luciferase reporter vector ,and analyze the microRNA(miRNA) which possibly regulate the expression of XIAP gene . Methods Polymerase chain reaction (PCR) was employed to amplify X IA P-3′UTR sequences from human cDNA ,in which luciferase reporter vector pGL3-Ctrl was inserted ,and the recombinant vector pGL3-Ctrl/XIAP was gained .Target Scan 6 .2 soft-ware was adopted to predict the miRNA which possibly combined with the X IA P-3′UTR .pGL3-Ctrl/XIAP recombinant plasmids and the miRNA were co-transfected into A549 cells ,and the X IA P-3′UTR-luciferase activity was measured .Results Confirmed by digestion and DNA sequencing ,the X IA P-3′UTR-luciferase reporter recombinant was successfully constructed .Prediction of miRNA target sites indicated that X IA P gene may be the target of miR-200b ,miR-200c and miR-429 .Compared with miRNA mim-ic ctrl group ,miR-200b ,miR-200c and miR-429 significantly reduced the luciferase activity of pGL 3-Ctrl/XIAP with statistically significant difference(P<0 .05) .Conclusion X IA P-3′UTR-luciferase reporter vector is successfully constructed .miR-200b ,miR-200c and miR-429 can obviously decrease the luciferase activity .
