ABSTRACT
Objective: To obtain purified and functional CDNF-his recombinant protein and prepare its polyclonal antibodies.Methods:Preparation of recombinant CDNF-his was carried out in HEK 293 T cells with pVR1012-CDNF-his successfully constructed transfected into them.Then,the recombinant protein was purified by Ni-NTA immunoaffinity chromatography.The purity was analyzed by SDS-PAGE and the protein′s identity was tested by Western blot.MTT was used to verify the biological function of the protein purified.New Zealand white rabbits were immunized with purified CDNF-his protein for preparation of polyclonal antibodies.Results:pVR1012-CDNF-his expressed successfully in HEK 293 T cells.The purity of protein was up to more than 90%after purification.MTT showed that CDNF-his was able to protect PC 12 cells from damage by 6-OHDA.The polyclonal antibody was detected at the end of animal immunizing process.Conclusion: A method to express and purify protein using HEK 293T cell and following Ni-NTA immunoaffinity chromatography has been built.CDNF-his with biological activity is obtained based that.Finally, polyclonal antibodies of CDNF were generated successfully.
ABSTRACT
A gene encoding NAD+-dependent sorbitol dehydrogenase (SDH) in peach fruit was cloned and expressed in Escherichia coli. Recombinant SDH protein with 6×His-tagged was localized exclusively in the cytoplasmic soluble fraction of E. coli when the strains were grown for 4-5 h at 37 ºC. Highly pure protein was isolated by Ni2+-resin chromatography with 150 mM imidazole in 50 mM Tris, pH 8.0, by elution. In order to ensure that the recombinant SDH could be used for further study, the fluorescence and ultraviolet spectrum of the recombinant SDH were detected. Recombinant SDH was confirmed to be capable of oxidizing sorbitol by enzymatic activity assay. The activity of the recombinant SDH was 2.73 U mg-1min-1, which was similar with that directly extracted from peach fruits. The activities of SDH extracted from the fruits in different periods (30, 60, 90 days after flowing) were 7.75, 5.95, 3.26 U mg-1min-1, respectively.
ABSTRACT
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.