RESUMEN
IFT46 is one of the important components of intraflagellar transport complex B in Chlamydomonas reinhardtii, and plays important roles in the assembly, movement and perception of ciliary. To study its functional mechanism, a GST-tagged and an MBP-tagged prokaryotic expression plasmid, pGEX-2T-ift46 and pMAL-C2X-ift46 were constructed, respectively, by inserting ift46 into the pGEX-2T and pMAL-C2X vector, and then transformed into Escherichia coli BL21 (DE3) for protein expression. SDS-PAGE (15%) analysis results showed that the molecular weights of the fusion protein GST-IFT46 and MBP-IFT46 were 70 kDa and 86 kDa, respectively. We used the fusion protein GST-IFT46 purified by affinity adsorption purification (more than 95% purity) for immunity to New Zealand white rabbits. The 5th immune serum was collected and the antibody titer was determined to be 256 000 by ELISA. The antiserum was purified by Protein A affinity adsorption purification and immobilized MBP-IFT46 purification, and the specificity of polyclonal antibodies was evaluated by Western blotting and immunofluorescence. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT46 in C. reinhardtii, and IFT46 was mainly concentrated at basal body regions and few localized along the entire length of the flagellum as punctuated dots, which will make a foundation to further study the mechanism of IFT46 in cilia related diseases such as obesity, diabetes and polycystic kidney disease.
Asunto(s)
Animales , Conejos , Proteínas Algáceas , Alergia e Inmunología , Anticuerpos , Química , Western Blotting , Chlamydomonas reinhardtii , Química , Genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Técnica del Anticuerpo Fluorescente , Péptidos y Proteínas de Señalización Intracelular , Alergia e Inmunología , Plásmidos , Proteínas Recombinantes de FusiónRESUMEN
The purpose of this study was to compare the efficiency of 3 different methods in purifying engineered anti-HBsFab. Anti-HBsFab Supernatants containing anti-HBsFab were prepared by ultrasound lysis of host cells of anti-HBsFab positive clone. Anti-Fab-sepharose gel, streptococcal protein G-sepharose gel (Prot G gel) and Ni-NTA-agarose gel were used to purify Fabs according to the reagents protocols. Then the concentrations of purified proteins were determined. SDS-PAGE was used for the measurement of purified Fabs'purity. HBsAg based ELISA was chosen to determine the Fabs' bio-activity. The results indicated that Fab recovery of different gels in equal volume were different. The recovery of Ni-NTA GEL was greater than that of Prot G gel, and the recovery of Prot G gel was greater than that of anti-Fab gel. Purity of the Fab isolated by 3 different gels was confirmed by SDS-PAGE. The binding capacity of anti-HBs Fab to HBsAg of these three gels had no signifticant difference. Our data suggested that each method had its advantage and usage limitation, Ni-NTA method demonstrated much better performance in economy, management, and efficiency over the other two methods.