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.
Artigo
em Chinês
| 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>
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Plasmídeos
/
Ligação Proteica
/
Biblioteca Gênica
/
Técnicas do Sistema de Duplo-Híbrido
/
Biologia Celular
/
Células Endoteliais
/
Proteínas Angiogênicas
/
Proliferação de Células
/
Proteína 1 Inibidora de Diferenciação
/
Genética
Tipo de estudo:
Estudo diagnóstico
/
Estudo de rastreamento
Limite:
Adulto
/
Humanos
Idioma:
Chinês
Revista:
Journal of Southern Medical University
Ano de publicação:
2007
Tipo de documento:
Artigo
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