Novel immunodominant epitopes derived from MAGE-A3 and its significance in serological diagnosis of gastric cancer

Abstract

Purpose: To evaluate the significance of MAGE-A3 novel immunodominant epitopes in serological diagnosis of gastric cancer.

Methods: B cell, CTL, and Th epitopes of MAGE-A3 were analyzed using computer-assisted techniques. Three possible immunodominant epitope peptides located at 5aa-23aa (QRSQHCKPEEGLEARGEAL), 112aa-131aa (KVAELVHFLLLKYRAREPVT), and 232aa-246aa (EGREDSILGDPKKLL) with potential B cell-dominant epitope, high-score HLA-A2 and A24 restriction CTL epitope, and HLA-DRB restriction Th epitope were selected. After optimized by prokaryotic codon, these genes were expressed as Trx-His-tag recombinant proteins in Escherichia coli and purified by Ni-NTA agarose beads. Three recombinant proteins were identified by Western blotting using His-tag monoclonal antibody and the serum antibodies from the patient of gastric cancer. The level of specific antibodies in the sera from 210 patients with gastric cancer, 56 patients with chronic gastritis, and 116 healthy controls was further analyzed by indirect ELISA.

Results: Three MAGE-A3 epitope recombinant proteins about 20 kDa molecular weight were specifically recognized by His-tag monoclonal antibody and the serum of gastric cancer patients. ELISA based on the epitope recombinant protein indicated that gastric cancer patients had significantly higher reactivity to these immunodominant epitope proteins compared with chronic gastritis and healthy individuals (P < 0.05). Furthermore, the serum antibody positive rate in the gastric cancer group was also significantly higher than that in the chronic gastritis patients and healthy controls (P < 0.05), while there was no significant difference in gastritis group and the healthy control group (P > 0.05).

Conclusions: These study results demonstrated that these three predictive epitopes may be potential targets for applications in the design of serological diagnosis tools for gastric cancer.

Fig. 1

Identification of MAGE-A3 immunodominant epitope recombinant plasmids by PCR and sequencing. a PCR analysis of pET32a/MAGE-A3 immunodominant epitope plasmid. Lane 1 BL21, Lane 2: pET32a(+) empty vector, Lane 3 pET32a/MAGE-A3 immunodominant epitope 1(5–23aa), Lane 4 pET32a/MAGE-A3 immunodominant epitope 2(112–131aa), Lane 5 pET32a/MAGE-A3 immunodominant epitope 3(232–246aa), M: λDNA/HindIII DNA Marker. b MAGE-A3 immunodominant epitope recombinant plasmid sequencing

Fig. 2

Expression, purification, and identification of MAGE-A3 immunodominant epitope recombinant proteins by SDS–PAGE and Western blot analysis. a MAGE-A3 immunodominant epitope recombinant protein expression detection by SDS–PAGE analysis; b MAGE-A3 immunodominant epitope recombinant protein expression detection by Western blotting assay using a monoclonal antibody against His protein. c Purification of MAGE-A3 immunodominant epitope recombinant protein assay by SDS–PAGE analysis. M: protein molecular weight standard, Lane 1 pET32a vector protein (Trx-His) at 19 kDa, Lane 2 MAGE-A3 immunodominant epitope 1 (TrX-His-epitope 1) at 20 kDa, Lane 3 MAGE-A3 immunodominant epitope 2 (TrX-His-epitope 2) at 20 kDa, Lane 4 MAGE-A3 immunodominant epitope 3 (TrX-His-epitope 1) at 20 kDa

Fig. 3

MAGE-A3-specific serum IgG antibody levels in mice following immunization ELISA based on the MAGE-A3 immunodominant epitope recombinant proteins and Trx-His protein analysis detected the serum IgG level in immunized BALB/C mice following on 0, 3, 5, 7, 9, and 12 weeks. The results showed that MAGE-A3 immunodominant epitope recombinant protein 1-, 2-, and 3-specific serum antibody levels increased following the immune weeks, reached the peak at week 7, and remained at this level at least until week 12

Fig. 4

Western blot analysis of MAGE-A3 immunodominant epitope proteins in the serum of gastric cancer patients. Serum from gastric cancer patients as the primary antibody; the purified MAGE-A3 immunodominant epitope proteins and the purified His-tag protein were detected by Western blot. Lane 1 Trx-His, Lane 2 MAGE-A3 immunodominant epitope 1, Lane 3 MAGE-A3 immunodominant epitope 2, Lane 4 MAGE-A3 immunodominant epitope 3

Fig. 5

ELISA analysis of MAGE-A3 immunodominant-epitope-protein-specific IgG antibodies in the serum. Serum-specific MAGE-A3 antibodies detected by ELISA analysis based on the MAGE-A3 immunodominant epitope protein in three groups of patients with gastric cancer, chronic gastritis, and healthy controls. *P < 0.05, compared with others

Fig. 6

The serum antibody positive detection rate of MAGE-A3 in gastric cancer patients; cutoff values of MAGE-A3 immunodominant epitopes 1-, 2-, and 3-specific IgG for the diagnosis of gastric were 0.517, 0.620, and 0.519, respectively; the results showed that the positive rate in the gastric cancer group was significantly higher than that in the chronic gastritis patients and healthy controls (P < 0.05), while there was no significant difference in gastritis group and the healthy control group (P > 0.05)