Prokaryotic Expression Of Human Regenerating Protein (REG1A) And Its Monoclonal Antibody

ObjectiveTo construct an vector for human regenerating protein(Reg1a)which is used to express His-Reg1 a fusion protein and prepare monoclonal antibodies of Reg1 a and the Reg1 a of human clinical samples are tested by the above antibodies.MethodsHis-tag was introduced at the 5’ end of Reg1 a gene by designing primers and PCR amplification.Then Reg1 a was combined to p ET32 a plasmid by homologous recombination.Gene sequencing analysis was performed to identify recombinant plasmid p ET32a-Reg1 a.The E.coli BL21(D3)which recombinant plasmid was transformed into are induced to express Reg1 a fusion protein at appropriate temperature and IPTG concentration.The Reg1 a protein was purified and isolated using a nickel agarose affinity chromatography column,and then Western blot was used to analysis specificity.Reg1 a fusion protein was used as an antigen to immunizing of mice.Splenic lymphocytes from immunized mice were taken and fused with myeloma cells.Hybridoma was screened to obtain cells that specifically secreted anti-human Reg1 a monoclonal antibody.The type and potency of monoclonal antibodies were identified by ELISA,and the specificity analysis of monoclonal antibodies was identified by Western blot.Finally,clinical samples were tested with hybridoma antibodies.ResultsThe His-Reg1 a gene sequence was successfully cloned and meanwhile the p ET32a-Reg1 a expression vector was constructed.It was clear that the recombinant protein existed in soluble form in the lysate supernatant of the E.coli,and the human Reg1 a protein was obtained with a purity of over 90%.After cell fusion,screening and subcloning,four stable and efficient hybridoma cell lines secreting specific antibodies were obtained.The antibodies of hybridoma were all Ig G1 type,and the light chain was κ type.The monoclonal antibody could specifically recognize and bind human Reg1 a protein in serum and tissues.ConclusionThe fusion protein of Reg1 a was successfully obtained,and the mouse-derived anti-human Reg1 a monoclonal antibody was prepared,and m Abs of Reg1 a were applied to the detection of clinical samples..