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

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.

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.

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.

The polyclonal antibody specifically recognizing IGF2BP3 has been successfully generated.