The Prokaryotic Expression And Application Research On Heart Fatty Acid Binding Protein

Heart-type fatty acid-binding protein (H-FABP), a 14-15KDa protein, is a lowmolecular weight protein which is abundantly present in heart tissue. H-FABPplays an important role in the intracellular transportation of fatty acid, themetabolism of lipids, regulating cell of acute myocardial infarction (AMI).In this study,the encoding sequence for mature peptide of human H-FABPwas amplified with RT-PCR and inserted into pQE30 vector to establish theprokaryotic expressing system.The recombinant plasmid (pQE-H-FABP) wastransformed into the competent cells of host strain of M15. Then expression of thetarget protein was induced with IPTG..The cloned fragment of human H-FABPwas 99.9% homology with that in genbank (NM004012), which was deduced toexpress 132 animo acids mature peptide of human H-FABP correctly. Theexpressed protein was 15kD in SDS-PAGE as we expected, was recognized by acommercial McAb(Clone 6B6) and was 27% in the total proteins. After G50chromatograph we obtain the target protein.The specific sequence of affinity ligand of H-FABP was obtained with thePh.D.-12 Phage Display Peptide Library Kit subsequently. After four round ofpanning, we obtain two ELISA positive sequences ofW-P-N-H-H-M-L-H-K-R-W-P and W-P-H-Q-K-L-H-L-M-R-H-S. Two peptideswere synthedized by manual way.After reverse phase HPLC(high pressure liquidchromatography) and mass spectrometry ,the target peptides coupled with pyreneas a fluorescent probes were detect with the samples of AMI patients. Theexperiment indicates that the samples of AMI patients are all potive. Kd valuedetermination of Peptide lˊ with AMI samples are beteen 37.9μM and 88.5μM..The resultreflects that the target ligand peptides we got have good effect on clinic application.The study makes it possible for application of the product.