Bacterial expression of 183-227aa region of HER3 extracellular domain I and preparation and identification of its polyclonal antibodies.
10.12122/j.issn.1673-4254.2020.06.06
- Author:
Lei ZHU
1
;
Pingchuan YUAN
1
;
Zhigang ZHAO
1
;
Xin WANG
1
;
Guodong WANG
1
;
Liang YAN
2
Author Information
1. Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Wuhu 241002, China.
2. Anhui Province Key Laboratory of Active Biological Macromolecules, Wuhu 241002, China.
- Publication Type:Journal Article
- Keywords:
HER3;
affinity chromatography;
dimerization;
fusion expression;
polyclonal antibody
- MeSH:
Animals;
Antibodies;
Enzyme-Linked Immunosorbent Assay;
Escherichia coli;
Humans;
Plasmids;
Rats;
Rats, Sprague-Dawley;
Receptor, ErbB-3;
immunology;
Recombinant Fusion Proteins
- From:
Journal of Southern Medical University
2020;40(6):806-813
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE: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.
METHODS: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 MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B.
RESULTS:The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG.
CONCLUSIONS:We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.
