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Preparation of Polyclonal Antibodies of Human Rbp4 and Its Preliminary Application

Author: SunYuXiu
Tutor: LuYunXia
School: Anhui Medical University,
Course: Biochemistry and Molecular Biology
Keywords: Human retinol binding protein 4 pET-28a () Inclusion bodies Nickel affinity chromatography Polyclonal antiserum
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 25
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Abstract


The purpose of preparation of anti- human retinol binding protein 4 (Retinal binding protein4, RBP4) polyclonal antiserum , initially applied to type 2 diabetes mellitus (T2DM) patients with serum RBP4 detection . From normal adult liver tissue extraction of total RNA , RT - PCR was amplified human RBP4 cDNA full-length , connected with pMD-18T Vector to construct the cloning vector pMD - hRBP4 . Design with BamH Ⅰ and Hind Ⅲ digestion sites upstream and downstream primers , PCR and restriction enzyme digestion to construct the prokaryotic expression vector pET28a ( ) - hRBP4 the calcium chloride law was transformed into BL21 (DE3), extracted plasmid digestion analysis and sequencing , the IPTG induction protein product identified western blotting . To optimize induced expression of conditions such as IPTG concentration , time and temperature , nickel affinity chromatography rhRBP4, immunize New Zealand white rabbits to prepare polyclonal antiserum , Western blotting was used to detect the specificity ELISA assay antibody titer . With polyclonal antiserum detection T2DM patients with serum RBP4 level . Results recombinant plasmid pET -28a ( ) - hRBP4 the the digestion results expected size completely consistent sequencing results confirmed that the resulting cDNA sequences in GenBank hRBP4 sequence is basically the same , and Western blotting results confirmed that the monoclonal antibody specific to the recombinant protein with hRBP4 binding. IPTG induction of optimal concentration is 0.04 mmol / L, the optimum time of 6 h , the optimum temperature of 37 ° C , up to 44% of the total bacterial protein expression of recombinant proteins , and the form of inclusion bodies . SDS-PAGE electrophoresis results showed that the recombinant protein was obtained by affinity chromatography purity of 96% . Measured by ELISA titer antiserum 1:512000 . Western blotting showed antiserum with molecular weight a 21kDa protein binding , the same size with natural RBP4 serum of patients with T2DM . Conclusion We successfully constructed and hRBP4 cDNA full-length prokaryotic expression vector and rhRBP4 protein , polyclonal antiserum may be initially used for the detection of RBP4 in the serum of patients with T2DM , further RBP4 functional studies and clinical mass serum samples RBP4 detection of the foundation .

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