Prokaryotic Expression, Puriitcation And Monoclonal Antibody, Polyclonal Antibody Preparation Of Human Heart Type Fatty Acid Binding Protein

The encoding sequence of human H-FABP was amplified with RT-PCR andinserted into plasmid pET28a to establish the prokaryotic expressing system. Thecompetent cells of host strain of BL21were transformed by the recombinant plasmidpET28a-H-FABP. The prokaryotic expression system pET28a for human H-FABP wasconstructed in E.coli BL21. The Expression of the recombinant protein was inducedwith IPTG. The molecular weight of recombinant H-FABP is15kD by SDS-PAGEanalysis and the protein concentration is0.48mg/mL after purified by Q-HP column.Western blotting was identified the recombinant protein by a commercial anti-H-FABPmAb.By use of B cell epitope prediction software online, we analyzed β-turn structure,surface antigen accessibility, protein flexibility, protein antigens, hydrophobicity and thelinear epitope of H-FABP. The regions with strong antigen specificity were chosen to beused for immunization along with recombinant H-FABP. Polyclonal antiserum wasraised in rabbits according to a novel immunization procedure. The IgG fractions ofPolyclonal antiserum were purified using Protein A-Sepharose。To remove antibodiesreacting to the tag protein and residual bacterial protein, the polyclonal antibodies ofH-FABP eluted from the Protein A column. These antibodies show high specificity andsensitivity against H-FABP in ELISA, western blot.Balb/c mice were immunized with purified H-FABP and B cell epitope peptide ofH-FABP. Splenocytes of the immunized mice were collected and fused with the mousemyeloma cell line Sp2/0cells. The hybridoma cells that secreted H-FABP mAb weresubcloned with limited dilution. Ascites induced from mice belly cavity is purified byHiTrap IgG Purification HP. Indirect enzyme-linked immunosorbent assay （ELISA）was used to evaluate the affinity and titers of the mAbs. The immunoglobulin （Ig）subtype of mAb was determined by testing subtype kit. Six specific hybridoma cell lines which can stably secreting anti-H-FABP mAb（IgG subtype） were obtained. Thetiters of the four mAbs in ascites were above1:256000and affinity mostly reach to9.02×10^-9. Western blot analysis showed that the mAb had specific combination withH-FABP which possessed biological activities. The mAbs against H-FABP withspecificity have been established successfully, which can provide a potential value forestablishing a new method to detect H-FABP.Specific anti-H-FABP polyclonal antibodies and monoclonal antibodies, whichhave been obtained in our laboratory, were used as capture antibody and detectionantibody respectively, meanwhile, recombination H-FABP obtained by prokaryoticexpression was used as detection antigen. Then the antibody pairing test was developed.After optimizing reaction conditions of double-antibody sandwich ELISA（DAS-ELISA）,26cases of AMI serum and34cases of normal human serum were detected andanalyzed. The pair bond3-H5—1-F10of anti-H-FABP was obtained and theDAS-ELISA for quantitative detection of H-FABP was established successfuly. So, thepair bond3-H5—1-F10obtained in our experiment is proved to be more specific toH-FABP protein, which will be the important foundation of H-FABP detection anddeveloping new H-FABP immunodiagnostic kit.