Bacterial expression of 183-227aa region of HER3 extracellular domain I and preparation and identification of its polyclonal antibodies

To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide. The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to