RESUMEN
The aim of this research is to prepare high quality polyclonal antibodies against Ecp, a recently identified leucine-zipper protein. The full length cDNA of ecp was amplified by PCR, cloned into pGEX-4T-1(His)6 and transformed into E. coli DH 5alpha. After induction with IPTG, the GST-Ecp fusion protein from the lysate was bound to glutathione-Sepharose 4B and digested with thrombin. The released Ecp protein was further purified through Ni-NTA affinity chromatography to homogeneity. A rabbit was immunized with the purified Ecp, and the antibody generated against Ecp was purified by affinity chromatography. The results of the Western blot showed that Ecp is present in various development stages of Drosophila melanogaster, from larvae to adult.
Asunto(s)
Animales , Conejos , Anticuerpos , Alergia e Inmunología , Metabolismo , Western Blotting , Cromatografía de Afinidad , Proteínas de Drosophila , Genética , Alergia e Inmunología , Drosophila melanogaster , Metabolismo , Electroforesis en Gel de Poliacrilamida , Regulación del Desarrollo de la Expresión Génica , Genética , Fisiología , Proteínas Fluorescentes Verdes , Genética , Metabolismo , Factores de Iniciación de Péptidos , Genética , Alergia e Inmunología , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión , Genética , Alergia e Inmunología , MetabolismoRESUMEN
A central portion of nifS, designated nifS', was amplified from Azotobacter vinelandii. The nifS' was cloned into pUC18 to make pUCS. Then pUCS was integrated into Azotobacter vindandii chromosome DNA by homologous recombination. This nifS disruption mutants were generated by single cross-over event and selected by Amp resistence on BBGN medium. The nifS disruption mutant (named SU1) was affirmed by southern blot and PCR amplification. SU1 grows rapidly on BBGN, but very slowly on Burk's N-free medium. This phenomenon showes that SU1 nearly lost its nitrogen fixation ability because of the disruption of nifS. The successful construction of SU1 is helpful for further research on the effect of nifS on the structure and function of nitrogenase component-Ⅰ and Ⅱ.