Screening of AGGF1 interacting with inhibitor of differentiation-1 / 南方医科大学学报
Journal of Southern Medical University
;
(12): 1379-1381, 2007.
Article
in Chinese
| WPRIM
| ID: wpr-283124
ABSTRACT
<p><b>OBJECTIVE</b>To screen the proteins interacting with inhibitor of differentiation 1(Id1) using yeast two-hybrid analysis in adult human lung cDNA libraries.</p><p><b>METHODS</b>The coding sequence of Id1 was amplified by PCR and cloned into the bait plasmid. The recombinant bait vector pHybLex/Zeo-Id1 was verified by restriction endonuclease digestion before transformation into the yeast strain EGY48/pSH18-34, which was tested subsequently for reporter genes Leu2 and LacZ activation. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into the yeast strains and screened to obtain Leu2(+) and Leu2(+)LacZ(+) clones, with the false positive clones excluded using positive and negative controls, and the plasmid of the true positive clone was sequenced and blasted for homological analysis.</p><p><b>RESULTS</b>Successful construction of pHybLex/Zeo-Id1 was confirmed by enzyme digestion. After transformation of pHybLex/Zeo-Id1 into EGY48/pSH18-34, no specific reporter genes Leu2 and LacZ activation was found. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into yeast strain, and 198 Leu(+) clones and 19 Leu(+)LacZ(+) double positive clones were obtained. After elimination of the false positive clones, one true positive clone was obtained, whose plasmid analysis by sequencing and blasting indicated high homology (99.5%, 556/559) to AGGF1 (an angiogenic factor with G-patch and FHA domains 1). AGGF1 expression was confirmed in the true positive yeast cells by Western blotting.</p><p><b>CONCLUSION</b>AGGF1 is confirmed to interact with Id1 by yeast two-hybrid analysis for screening adult human lung cDNA libraries.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Protein Binding
/
Gene Library
/
Two-Hybrid System Techniques
/
Cell Biology
/
Endothelial Cells
/
Angiogenic Proteins
/
Cell Proliferation
/
Inhibitor of Differentiation Protein 1
/
Genetics
Type of study:
Diagnostic study
/
Screening study
Limits:
Adult
/
Humans
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2007
Type:
Article
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