Cloning of human migfilin N-terminal domain and preparation of anti-migfilin polyclonal antibody / 南方医科大学学报
Journal of Southern Medical University
; (12): 915-918, 2008.
Article
en Zh
| WPRIM
| ID: wpr-280069
Biblioteca responsable:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To clone migfilin-N terminal sequence into E.coli and obtain a fusion protein for preparing rabbit polyclonal antibody against migfilin, thereby facilitating the study of the role of migfilin in the biological behavior of colon cancer.</p><p><b>METHODS</b>Based on human migfilin cDNA sequence, a pair of primers was designed to amplify migfilin-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-1 with EcoRI/XhoI sites, and the target recombinant plasmids were identified with enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL(21) cells, the GST-migfilin-N fusion protein was expressed with IPTG induction. Glutathione-sepharose beads were used to purify the fusion protein, and anti-migfilin polyclonal antibody was produced by immunization of rabbits with the purified GST-migfilin N-terminal fusion protein. The resultant anti-migfilin polyclonal antibody was purified by protein A beads and used for Western blotting for detecting migfilin expression in different cell lines.</p><p><b>RESULTS AND CONCLUSION</b>The migfilin-N terminal gene fragment was cloned successfully, and purified GST-migfilin N-terminal fusion protein and anti-rabbit migfilin polyclonal antibodies were obtained. Western blot analysis demonstrates that the antibodies specifically detected migfilin expression in the cell lines, which may facilitate further investigation of the role of migfilin in the biology of colon cancer.</p>
Texto completo:
1
Base de datos:
WPRIM
Asunto principal:
Patología
/
Proteínas Recombinantes de Fusión
/
Datos de Secuencia Molecular
/
Secuencia de Bases
/
Moléculas de Adhesión Celular
/
Química
/
Western Blotting
/
Clonación Molecular
/
Análisis de Secuencia de ADN
/
Neoplasias del Colon
Límite:
Animals
/
Humans
Idioma:
Zh
Revista:
Journal of Southern Medical University
Año:
2008
Tipo del documento:
Article