Prokaryotic expression and preparation of antibody of human neutrophil gelatinase-associated lipocalin

Abstract:

Objective：To obtain human neutrophil gelatinase-associated lipocalin（NGAL） recombinant protein and its polyclonal and monoclonal antibodies，and to provide a powerful experimental basis for further functional research and clinical application of NGAL. Methods：The coding region of NGAL mature peptide gene was codon-optimized，chemically synthesized，and then inserted into the prokaryotic expression vector pW28. Transfected E.coli B834 cells were induced by IPTG to produce rhNGAL. The recombinant pro－tein was was purified by Ni2+-NTA affinity chromatography column. The aggregation in solution was analyzed by Hiload Superdex 75 gel flittration column. Rabbits and BALB/c mice were immunized with the rhNGAL to produce polyclonalant antibodies and high speci－ficity monoclonal antibodies by hybridoma technology to identify antibody subtypes. After being purified by Protein G affinity column，the antibodies was detected by Isothermal Titration Calorimetry，non-competitive ELISA and SDS-PAGE analysis. Finally，Western blot，immunofluorescence and immunohistochemistry were used for antibody identification. Results：NGAL recombinant protein was successfully expressed with high purity and mainly existed in monomer form. Recombinant procalcitonin was successfully expressed. Rabbit polyclonal antibody was also produced. Six hybridoma cell lines stably secreting mAbs were screened，hereinto the mAb25 was IgG1 subtype，and the others belonged to IgG2a subtype. The affinity constant of mAb19 and 35 was 3.06×109 and 2.14×109，respectively. The SDS-PAGE analysis showed that the purity was higher than 90%. Excellent immuno-reactivity and specificity of the antibodies was showed by Western blot immunofluorescence and immunohistochemistry analysis. Conclusion：Highly purified rhNGAL protein，anti-NGAL polyclonal antibodies and specific monoclonal antibodies are obtained in this study. The findings lay the foundation for the further study on the function of NGAL in pathophysiology and other regions，and promotes the development of domestic NGAL clinical diagnostic reagents.