Preparation Of Polyclonal Antibodies Of Human Rbp4 And Its Preliminary Application

Objective: To prepare the rabbit polyclonal antibodies against human RBP4 (retinol binding protein 4) and apply it to assay serum RBP4 level in type 2 diabetic patients.Method: Total RNA was isolated from normal human liver, then RBP4 were amplified by two-step RT-PCR. The cloning plasmid pMD-hRBP4 was constructed after retrieval of the amplification products, then RBP4 was subcloned with primers containing restriction endonucleases recognition sites of BamHâ… and Hindâ…¢, ligated into pET-28a(+), transducted into BL21(DE3). After identified with double-enzyme cutting and sequencing, the bacteria transformed with pET-28a(+)-hRBP4 were induced with IPTG and expressed proteins were identified with western blotting. Then induction conditions of highly efficient expression were experimented and the fusion protein expressed in E. coli following IPTG induction was purified by Ni2+ affinity chromatography. The purity of the recombinant protein was detected with SDS-PAGE. The rabbit was immunized with the purified fusion protein for 3 times.The specificity and sensitivity were detected by ELISA. RBP4 in serum of type 2 diabetic patient was assayed by western blotting with rabbit polyclonal antiserum.Result: The sequencing result proved that the cloned hRBP4 cDNA sequence was uniform with its sequence stored in Genbank. The double enzyme cutting result of recombinant plasmid pET-28a(+)- hRBP4 was coincident with expecting. RBP4 was highly expressed in the form of insoluble inclusion body in E.Coli. Western blotting result showed that the highly expressed protein could bind with hRBP4 monoclonal antibody specifically. The best induction concentration of IPTG was 0.04 mmol/L, the best induction time was 6h and the best induction temperature was 37℃. The result of SDS-PAGE indicated that the purity of rhRBP4 could be reached 96% after Ni²⁺ affinity chromatography.The polyclonal antiserum obtained by immunization of New Zealand white rabbit with rhRBP4 showed a serum antibody titer of 1:512000. Western blotting analysis proved that the rabbit polyclonal antiserum could specifically recognize 21kDa protein which is as large as natural RBP4 protein.Conclusion: The prokaryotic expression plasmids and corresponding purified rhRBP4 were obtained, the rabbit polyclonal antiserum against hRBP4 could specifically recognize natural protein expressed in serum of patient with Type 2 Diabetes Mellitus, this result indicated that it can be used for further study and clinical large scale detection of human RBP4 in serum or tissues.