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Expression, immunogenicity and clinical significance analysis of thyroid‑stimulating hormone receptor fusion proteins

  • Authors:
    • Xia Chen
    • Hui Chen
  • View Affiliations / Copyright

    Affiliations: Department of Endocrinology and Metabolism, The Second Hospital and Clinical Medical School, Lanzhou University, Lanzhou, Gansu 730000, P.R. China
    Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 274
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    Published online on: July 29, 2025
       https://doi.org/10.3892/mmr.2025.13639
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Abstract

Thyroid function is regulated in a substantial manner by thyroid‑stimulating hormone receptor (TSHR), and aberrant alterations in thyroid function are triggered by the interaction of TSHR with its antibodies, thyroid‑stimulating hormone receptor antibodies (TRAb). The expression, immunogenicity and clinical significance of fusion proteins comprising different structural domains of TSHR were investigated. Fusion proteins containing several human TSHR (hTSHR) structural domains were created. In vitro experiments utilized these fusion proteins as antigens to specifically bind and analyze patient sera using an ELISA. To investigate the immunogenicity and clinical significance of various structural domains of TSHR, in vivo experiments included immunizing BALB/c mice with various fusion proteins of hTSHR, measuring serum autoantibodies, assessing thyroid function, performing histological examination and using flow cytometry to identify changes in T cell subsets. Three distinct hTSHR fusion protein fragments (hTSHR289, hTSHR290 and hTSHR410) were synthesized. The hTSHR290 fusion protein demonstrated the highest binding reaction with TRAb+ sera from patients with hypothyroidism, and the hTSHR289 fusion protein demonstrated considerable specific binding reactivity with stimulating antibodies, as observed in sera from patients with hyperthyroidism. Pathological alterations associated with hyperthyroidism were observed in mice in the hTSHR289 fusion protein group, while pathological changes associated with hypothyroidism were observed in mice in the hTSHR290 fusion protein group. Immunized BALB/c mice exhibited increased levels of CD4+ T cell subsets, and decreased levels of CD8+CD122+ and CD4+CD25+ T cell subsets. Fusion proteins of different structural domains of TSHR exhibited varying immunogenicity. The hTSHR289 fusion protein and hTSHR290 fusion protein prepared in the present study could serve as a basis for the development of ELISA kits for the detection of thyroid‑stimulating immunoglobulins and TSHR‑blocking antibodies. Fusion proteins of different structural domains of TSHR induced clinical symptoms of hyperthyroidism and hypothyroidism in mice. The present study provides a scientific basis for future studies on the etiology and mechanisms of autoimmune thyroid diseases, as well as the invention of novel methods for TRAb detection.
View Figures

Figure 1

Expression of different fusion
proteins of hTSHR. (A) Sequence of different fusion proteins of
hTSHR. hTSHR410 (amino acid sequence 21–410), hTSHR289 (amino acid
sequence 1–289) and hTSHR290 (amino acid sequence 290–410). (B)
Restriction fragments on an agarose gel. Lane 1 shows hTSHR289,
lane 2 shows hTSHR290 and lane 3 shows hTSHR410. (C) SDS-PAGE of
purified hTSHR289. Lanes 1–3 show different imidazole-purified
proteins (250, 350 and 150 mmol/ml, respectively) and lane 4 is
unpurified fusion protein after fragmentation. (D) hTSHR410 fusion
protein. Lane 1 is the unpurified fusion protein after
fragmentation and lanes 2–4 show different imidazole-purified
proteins (150, 250 and 350 mmol/ml, respectively). (E) hTSHR290
fusion protein. Lane 1 is the unpurified fusion protein after
fragmentation and lanes 2–4 show different imidazole-purified
proteins (150, 250 and 350 mmol/ml, respectively). hTSHR, human
thyroid-stimulating hormone receptor.

Figure 2

Binding of different fusion proteins
with TSHR antibodies and His antibodies. (A) Binding with TSHR
antibodies. hTSHR290 shows bands at 39 kDa (lane 1); blank (lane 2)
hTSHR289 shows bands at 47 kDa (lanes 3 and 4); hTSHR410 shows
bands at 70 kDa (lanes 5–8). (B) Binding with His antibodies.
hTSHR289 shows bands at 47 kDa (lanes 1 and 2); hTSHR290 shows
bands at 39 kDa (lane 3); hTSHR410 shows bands at 70 kDa (lane 4).
His, histidine; hTSHR, human thyroid-stimulating hormone
receptor.

Figure 3

Specific binding of different fusion
proteins as antigens with antibodies. Binding of fusion proteins
from the hTSHR290, hTSHR289 and hTSHR410 groups with sera from
different groups of patients. *P<0.05, **P<0.01. One-way
ANOVA with Tukey's post hoc test was used for comparisons of
multiple groups of parametric data. hTSHR, human
thyroid-stimulating hormone receptor; TRAb, thyroid-stimulating
hormone receptor antibodies.

Figure 4

Changes in mouse thyroid function.
Measurements of post-immunization levels of (A) T4, (B) TRAb and
(C) TSH in mice. *P<0.05, **P<0.01. One-way ANOVA with
Tukey's post hoc test was used for comparisons of multiple groups
of parametric data. hTSHR, human thyroid-stimulating hormone
receptor; T4, thyroxine; TRAb, thyroid-stimulating hormone receptor
antibodies; TSH, thyroid-stimulating hormone.

Figure 5

Pathological changes in mouse thyroid
tissues. Representative images of pathological changes in the
thyroid glands of mice from the hTSHR289 group after immunization
following H&E staining at a magnification of (A) ×20 and (B)
×40. Arrows indicate diffuse hyperplasia with an increase in
follicular epithelial cells and papillary protrusions Schematic
representation of pathological changes in the thyroid glands of
mice from the hTSHR290 group after immunization following H&E
staining at a magnification of (C) ×20 and (D) ×40. Arrows indicate
high columnar cells with destroyed and atrophied follicles
accompanies by extruded colloids. Schematic representation of
pathological changes in the thyroid glands of mice from the
hTSHR410 group after immunization following H&E staining at a
magnification of (E) ×20 and (F) ×40. Arrows indicate high columnar
cells, follicular epithelial cell proliferation and fibrous tissue
increase. Schematic representation of pathological changes in the
thyroid glands of mice from the control group after immunization
following H&E staining at a magnification of (G) ×20 and (H)
×40. hTSHR, human thyroid-stimulating hormone receptor.

Figure 6

Changes in T cell subsets in mice
after immunization. (A) Flow cytometry analysis of changes in
CD4+ T cells in the hTSHR289, hTSHR290 and hTSHR410
groups after immunization. (B) Flow cytometry analysis of changes
in CD8+ T cells in the hTSHR289, hTSHR290 and hTSHR410
groups after immunization. *P<0.05 (One-way ANOVA with Tukey's
post hoc test). hTSHR, human thyroid-stimulating hormone receptor;
ns, not significant.

Figure 7

Changes of specific regulatory T cell
subsets in mice after immunization. (A) Flow cytometry analysis of
changes in CD4+CD25+ T cells in the hTSHR289,
hTSHR290 and hTSHR410 groups after immunization. (B) Flow cytometry
analysis of changes in CD8+CD122+ T cells in
the hTSHR289, hTSHR290 and hTSHR410 groups after immunization.
*P<0.05 (One-way ANOVA with Tukey's post hoc test. hTSHR, human
thyroid-stimulating hormone receptor.
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Copy and paste a formatted citation
Spandidos Publications style
Chen X and Chen H: Expression, immunogenicity and clinical significance analysis of thyroid‑stimulating hormone receptor fusion proteins. Mol Med Rep 32: 274, 2025.
APA
Chen, X., & Chen, H. (2025). Expression, immunogenicity and clinical significance analysis of thyroid‑stimulating hormone receptor fusion proteins. Molecular Medicine Reports, 32, 274. https://doi.org/10.3892/mmr.2025.13639
MLA
Chen, X., Chen, H."Expression, immunogenicity and clinical significance analysis of thyroid‑stimulating hormone receptor fusion proteins". Molecular Medicine Reports 32.4 (2025): 274.
Chicago
Chen, X., Chen, H."Expression, immunogenicity and clinical significance analysis of thyroid‑stimulating hormone receptor fusion proteins". Molecular Medicine Reports 32, no. 4 (2025): 274. https://doi.org/10.3892/mmr.2025.13639
Copy and paste a formatted citation
x
Spandidos Publications style
Chen X and Chen H: Expression, immunogenicity and clinical significance analysis of thyroid‑stimulating hormone receptor fusion proteins. Mol Med Rep 32: 274, 2025.
APA
Chen, X., & Chen, H. (2025). Expression, immunogenicity and clinical significance analysis of thyroid‑stimulating hormone receptor fusion proteins. Molecular Medicine Reports, 32, 274. https://doi.org/10.3892/mmr.2025.13639
MLA
Chen, X., Chen, H."Expression, immunogenicity and clinical significance analysis of thyroid‑stimulating hormone receptor fusion proteins". Molecular Medicine Reports 32.4 (2025): 274.
Chicago
Chen, X., Chen, H."Expression, immunogenicity and clinical significance analysis of thyroid‑stimulating hormone receptor fusion proteins". Molecular Medicine Reports 32, no. 4 (2025): 274. https://doi.org/10.3892/mmr.2025.13639
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