[Preparation and identification of the polyclonal antibody against human insulin-like growth factor 2 mRNA-binding protein 3].

Objective: To construct the prokaryotic expression vector of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), express and purify IGF2BP3 protein in E.coli, and prepare the polyclonal antibody against IGF2BP3.

Methods: The full open reading frame (ORF) of human IGF2BP3 was amplified by PCR, subcloned into pET-28a vector, and transformed into E.coli BL21(DE3), in which expression of the His-tagged IGF2BP3 protein was induced by IPTG. This protein was subsequently purified by Ni-NTA purification system, refolded by removal of urea from the solution. BALB/c mice were immunized with the purified IGF2BP3 protein to produce polyclonal antibody against IGF2BP3. The resulting anti-sera were further characterized by ELISA, Western blotting and immunohistochemistry.

Results: IGF2BP3 gene we amplified was consistent with the sequence reported by GenBank. Prokaryotic expression vector pET-28a-IGF2BP3 was constructed. His-tagged IGF2BP3 protein was successfully expressed in BL21 (DE3) with relative molecular mass (Mr) about 70 000 after IPTG induction. After purified by Ni-NTA resin, the purity of the protein reached above 90%. After immunization, the titer of the IGF2BP3 mouse anti-serum was over 1:50 000 as determined by ELISA. Further, Western blotting and immunohistochemistry showed that the IGF2BP3 antibody could specifically recognize the target protein.

Conclusion: The polyclonal antibody specifically recognizing IGF2BP3 has been successfully generated.