Identification of the interactions between the truncated fragments of macrophage migration inhibitory factor and CD74 using a yeast two-hybrid system / 南方医科大学学报
Journal of Southern Medical University
; (12): 2383-2390, 2009.
Article
em Zh
| WPRIM
| ID: wpr-325111
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the interaction domains between macrophage migration inhibitory factors (MIF) and the extracellular segment of type-II trans-membrane protein CD74 using a yeast two-hybrid system.</p><p><b>METHODS</b>By using molecular cloning techniques, the DNA fragments encoding MIF, MIF(50-65) and MIF(1-50/65-115) were introduced into the pGBKT7 vector to construct the corresponding recombinant bait plasmids, and the DNA fragments encoding CD74(73-232), CD74(73-109), CD74(1109-149) and CD74(149-232) into the pGADT7 vector to construct the recombinant activation domain (AD) plasmids. PEG/LiAC method was employed to transform the above 3 recombinant bait plasmids paired with each of the 4 recombinant AD plasmids into the chemical competent yeast AH109 cells. The transformed yeast AH109 cells were screened consecutively on SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-alpha-gal nutritional media.</p><p><b>RESULTS</b>The results of restriction endonuclease digestion and DNA sequencing verified the correct construction of all the recombinant plasmids. The yeast AH109 cells transformed with each of the 3 recombinant bait plasmids could grow on SD/-trp nutritional media without autonomous activation effect on the reporter gene MEL1. The cells transformed with each of the 4 recombinant AD plasmids could also grow on SD/-leu nutritional media without activation of the reporter gene MEL1. Only the yeast AH109 cells co-transformed with MIF, MIF(50-65), or MIF(1-50/65-115) plasmid and CD74(73-232) plasmid could grow on SD/-Trp-Leu-Ade-His nutritional media with transcription activation of the reporter gene MEL1.</p><p><b>CONCLUSION</b>MIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).</p>
Texto completo:
1
Base de dados:
WPRIM
Assunto principal:
Fragmentos de Peptídeos
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Proteínas Recombinantes
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Antígenos de Diferenciação de Linfócitos B
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Antígenos de Histocompatibilidade Classe II
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Fatores Inibidores da Migração de Macrófagos
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Clonagem Molecular
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Técnicas do Sistema de Duplo-Híbrido
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Escherichia coli
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Matriz Extracelular
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Domínios e Motivos de Interação entre Proteínas
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Idioma:
Zh
Revista:
Journal of Southern Medical University
Ano de publicação:
2009
Tipo de documento:
Article