HER2 dimer steric hindrance avoiding and HER2 dual-targeting fusion protein and application thereof in HER2 immunohistochemical detection

Abstract

The invention discloses a fusion protein avoiding HER2 dimer steric hindrance and dual-targeting HER2 and application of the fusion protein in HER2 immunohistochemical detection, the fusion protein is formed by fusing Nb and Aby through a flexible connecting peptide G4S, the amino acid sequence table of the fusion protein is as shown in SEQ ID NO: 1, and the nucleotide sequence table of a coding gene of the fusion protein is as shown in SEQ ID NO: 2; wherein Nby and Aby respectively target a region I and a region III of an HER2 extracellular domain, and are prevented from being combined with a region II of a HER2 dimerization key region. Experimental results show that in breast cancer HER2 immunohistochemical detection, the fusion protein can perform double targeting on an HER2 extracellular domain I region and an HER2 extracellular domain III region through a double-site recognition mechanism, the steric hindrance of an HER2 dimer binding region is effectively avoided, meanwhile, monomers and dimers of HER2 in tissues can be recognized, and the overall detection sensitivity is remarkably improved. In addition, the fusion protein has a certain correlation trend with tumor invasiveness indexes, so that the fusion protein is expected to provide an effective basis for accurate screening of HER2 targeted therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection technology, and specifically relates to a fusion protein that avoids the steric hindrance of HER2 dimers and dual-targets HER2, and its application in HER2 immunohistochemical detection. Background Art

[0002] Human epidermal growth factor receptor 2 (HER2) is a key target for breast cancer (BC) treatment. Its overexpression is closely associated with increased tumor aggressiveness and poor prognosis. Currently, HER2 immunohistochemistry (IHC) and in situ hybridization (ISH) are the gold standard for clinical assessment of HER2 status. IHC is widely used due to its ease of use and low cost. However, HER2 exists in both monomeric and dimeric states. Traditional antibodies, due to their large molecular weight (150 kDa), have binding sites located in the same position. These binding regions may overlap with the HER2 dimerization site (ECD II or adjacent to it), resulting in steric hindrance and ineffective detection of dimerized HER2. This can lead to false-negative HER2 diagnoses or low IHC scores. Studies have shown that approximately 48% of samples with an IHC HER2 score of 0 actually have detectable HER2 expression, and the concordance between different pathologists for scoring low HER2 expression (1+ / 2+) samples is less than 26%, seriously impacting treatment decisions (e.g., the applicability of T-DXd).

[0003] In recent years, breakthroughs have been made in targeted therapies for breast cancers with low HER2 expression (such as trastuzumab deruxtecan, T-DXd). However, their efficacy is highly dependent on the accuracy of HER2 detection. Existing IHC detection antibodies, due to their large molecular weight and targeting the HER2 ECD IV region or transmembrane domain, are susceptible to interference from dimerization conformation and may only detect free monomeric HER2, leading to missed detection of activated dimers. Furthermore, the incomplete correspondence between HER2 gene amplification and protein expression (approximately 10% difference) further complicates diagnosis, necessitating the development of highly sensitive detection technologies.

[0004] To overcome these limitations, researchers have attempted to improve detection sensitivity through the use of multi-epitope antibodies or nanoprobes. However, existing technical solutions still face the following challenges:

[0005] (1) Steric hindrance is not resolved: the target region of most antibodies is still adjacent to the dimerization site, making it impossible to distinguish between monomeric and dimeric HER2;

[0006] (2) The probe is too large: Although ferritin (HFn)-based nanoparticles can carry multiple targeting molecules, their size (~12 nm) leads to poor tissue penetration. In addition, if the design is inexperienced, the detection label may be easily obscured, reducing the detection efficiency.

[0007] (3) Difficulty in clinical transformation: Complex preparation process and high cost limit its large-scale application.

[0008] Therefore, developing a detection tool that can specifically bind to the non-dimerization region of HER2, dual-target or multi-target HER2, have a molecular weight small enough to overcome the steric hindrance effect, and have both high sensitivity and clinical practicality is of great significance for achieving accurate breast cancer classification and personalized treatment. Summary of the Invention

[0009] In response to the technical problems in the background art, the present invention aims to provide a fusion protein that avoids steric hindrance of HER2 dimers and dual-targets HER2, and its use in HER2 immunohistochemical detection. In HER2 immunohistochemical detection of breast cancer, this fusion protein can specifically bind to the non-dimerized region of HER2, avoiding the HER2 dimer-binding region, thus resolving the steric hindrance problem caused by HER2 dimerization and significantly improving detection sensitivity, especially for the diagnostic accuracy of breast cancer samples with low HER2 expression (0 / 1+).

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] The first aspect of the present invention provides a fusion protein that avoids the steric hindrance of HER2 dimers and dual-targets HER2. The fusion protein is composed of a nanobody (Nby) and an affibody (Aby) fused through a flexible connecting peptide G4S, and is referred to as an Nby-Aby fusion protein. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO: 2; wherein, the Nby targets the HER2 extracellular region (ECD) I region (PBD database number: 5MY6_B), and the Aby targets the HER2 extracellular region (ECD) III region (GenBank: AEN27854.1).

[0012] Preferably, the binding regions of the nanobody (Nby) and affibody (Aby) are independent of the key region for HER2 dimerization (extracellular region (ECD) II), thereby effectively avoiding the steric hindrance of HER2 dimers.

[0013] Preferably, the N-terminus of the Nby-Aby fusion protein contains an HA tag to facilitate detection signal amplification.

[0014] Preferably, the amino acid sequence of the flexible connecting peptide (G4S) is Gly-Gly-Gly-Gly-Ser to ensure the independent targeting functions of Nby and Aby.

[0015] The second aspect of the present invention provides a method for preparing the above-mentioned fusion protein that avoids steric hindrance of HER2 dimers and dual-targets HER2, comprising the following steps:

[0016] S1. Synthesize the gene sequences of nanobody (Nby) and affibody (Aby) respectively, and construct the fusion gene by introducing the flexible connecting peptide G4S;

[0017] S2. Clone the fusion gene into the pET21a(+) expression vector and transform it into the E. coli BL21(DE3) host strain;

[0018] S3. IPTG (isopropyl-β-D-thiogalactopyranoside) was used to induce host bacteria expression, and the fusion protein was obtained by Ni-NTA chromatography purification, and the purity of the obtained fusion protein was verified by SDS-PAGE.

[0019] Preferably, the host bacteria induction conditions are: 37° C., 200 rpm shaking culture for 6 h, and a final IPTG concentration of 1 mM.

[0020] Preferably, the Ni-NTA chromatography purification uses 50-100 mM imidazole for elution and PBS with a pH of 7.4 as the dialysate.

[0021] The third aspect of the present invention provides the use of the above fusion protein in HER2 immunohistochemical detection.

[0022] Preferably, the method for detecting HER2 immunohistochemistry using the above fusion protein comprises the following steps:

[0023] A1. After dewaxing and antigen retrieval, breast cancer tissue sections were incubated with fusion protein at a working concentration of 200 μg / mL at 4°C overnight or at 37°C for 0.5-2 hours.

[0024] A2, signal amplification using rabbit anti-HA monoclonal antibody and HRP-labeled secondary antibody;

[0025] A3. After DAB color development, score the test results according to the guidelines.

[0026] Preferably, the method for detecting HER2 immunohistochemistry using the above fusion protein comprises the following steps:

[0027] B1. Label the fusion protein with HRP to obtain Nby-Aby-HRP fusion protein, then dialyze and measure the protein concentration;

[0028] B2. The TMA chip containing breast cancer tissue was dewaxed and hydrated, and then blocked with 3% H2O2 to block endogenous peroxidase and 5% goat serum (Beyotime) to block nonspecific binding. Finally, it was incubated with Nby-Aby-HRP fusion protein at a working concentration of 200 μg / mL at 4°C overnight, washed, developed with DAB, counterstained with hematoxylin, and mounted for microscopic examination.

[0029] A fourth aspect of the present invention provides a HER2 immunohistochemistry detection kit, which comprises the above-mentioned fusion protein, DAB color development solution and HA tag antibody.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention connects and fuses a nanobody (Nby) and an affibody (Aby) through a flexible connecting peptide (G4S) to construct a fusion protein. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding the fusion protein is shown in SEQ ID NO: 2. In the immunohistochemical detection of HER2 in breast cancer, the nanobody (Nby) and the affibody (Aby) in the fusion protein can target the HER2 extracellular domain region I and region III, respectively, and dual-target the HER2 extracellular domain region I and region III through a dual-site recognition mechanism, which not only effectively avoids the steric hindrance of the HER2 dimer, but also can identify the monomer and dimer of HER2 in the tissue, thereby improving the overall detection sensitivity.

[0032] (2) The present invention provides a fusion protein that avoids the steric hindrance of HER2 dimers and dual-targets HER2. It is predicted that its molecular weight is relatively small (23 kDa), which can effectively improve its permeability in tissues, and the HA tag is fully exposed. The signal amplification efficiency used in immunohistochemical detection is significantly better than that of HER2-targeted nanoparticles (such as Nby-HFn), and therefore has high applicability in clinical diagnosis.

[0033] (3) The fusion protein provided by the present invention, which avoids the steric hindrance of HER2 dimers and dual-targets HER2, shows a certain correlation trend with tumor invasiveness indicators (such as Ki67>30%, tumor size ≤2cm), suggesting that it is expected to provide an effective basis for the precise screening of HER2 targeted therapies such as T-DXd, and provides a new idea for the personalized selection of tumor targeted treatment plans, and therefore has a high therapeutic guidance value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 :A is the result of enzyme digestion identification of recombinant plasmid; (B) the peak diagram of plasmid construction and sequencing;

[0036] Figure 2 : A is the SDS-PAGE verification results of prokaryotic induced expression (lane 2) and protein purification (lane 3) of Nby-Aby;

[0037] Figure 3 The results are a comparison of the sensitivity of Nby-Aby and traditional antibodies in IHC detection;

[0038] Figure 4 Figure 3 Re-evaluation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) scores using Nby-Aby and the proposed mechanism for score improvement: (A) Tissue initially diagnosed with a HER2 score of 0 using conventional antibodies (con-Ab) was re-evaluated as HER2 2+ or 3+ using Nby-Aby; (B) Tissue initially diagnosed with a HER2 score of 1+ using conventional antibodies was re-evaluated as HER2 2+ or 3+ using Nby-Aby; (C) Tissue initially diagnosed with a HER2 score of 2+ using conventional antibodies was re-evaluated as HER2 2+ or 3+ using Nby-Aby; (D) Tissue initially diagnosed with a HER2 score of 3+ using conventional antibodies was re-evaluated as HER2 3+ using Nby-Aby; (E) Schematic diagram of the potential mechanism by which Nby-Aby improves HER2 immunohistochemistry scores compared with conventional antibodies. DETAILED DESCRIPTION

[0039] In the following description, specific details such as specific system structures and technologies are provided for the purpose of illustration rather than limitation to facilitate a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0040] Example 1

[0041] Preparation, expression and purification of Nby-Aby fusion protein

[0042] S1. Construction of recombinant plasmid:

[0043] The gene sequences of the nanobody Nby (targeting the HER2 ECD I region, PDB: 5MY6_B) and the affibody Aby (targeting the HER2 ECDIII region, GenBank: AEN27854.1) were synthesized. The nanobody Nby gene and the affibody Aby gene were fused via a G4S connecting peptide (sequence: Gly-Gly-Gly-Gly-Ser) to construct the fusion gene Nby-Aby. Figure 1 A.

[0044] Finally, the fusion gene Nby-Aby was cloned into the pET21a(+) expression vector (purchased from Thermo Fisher) to construct the recombinant plasmid, and double enzyme digestion was performed using NdeI and XhoI restriction enzymes (Takara) for verification. Figure 1 (A) and Figure 1 (B).

[0045] S2. Protein induction expression

[0046] The recombinant plasmid was transformed into E. coli BL21 (DE3) (purchased from Novagen), inoculated into LB medium containing 100 μg / mL ampicillin, and cultured at 37°C with shaking until OD600 = 0.6. 1 mM IPTG (Sigma) was then added, and expression was induced at 37°C for 6 hours. The cells were then collected by centrifugation.

[0047] S3. Protein purification and characterization

[0048] After the bacteria were lysed by ultrasonication, the supernatant was purified by Ni-NTA affinity chromatography (Qiagen) using PBS (pH 7.4) containing 50 mM imidazole as the eluent.

[0049] The purified fusion protein was analyzed by SDS-PAGE. Figure 2 . Figure 2 The results showed a single target band (23 kDa);

[0050] The HER2 scores of tissue microarray (TMA) samples in the following experimental examples were statistically correlated with ISH, Ki67, and tumor size using Fisher's exact test (SPSS 22.0).

[0051] Test Example 1

[0052] 1. The prepared Nby-Aby fusion protein was used for HER2 immunohistochemical detection. The specific steps are as follows:

[0053] (1) Tissue sample processing: 100 formalin-fixed paraffin-embedded (FFPE) breast cancer tissue sections (purchased from Outdo Biotech) with a thickness of 4 μm were selected and sequentially dewaxed and antigen retrieval (pH 6.0 sodium citrate buffer, incubated at 95°C for 20 minutes). The experimental conditions of this process must be strictly controlled, otherwise it may lead to destruction or insufficient exposure of the antigen epitope.

[0054] (2) Incubation of Nby-Aby fusion protein and signal amplification using rabbit anti-HA monoclonal antibody and HRP-labeled secondary antibody: a) Sections were blocked with 3% H2O2 to block endogenous peroxidase and 5% goat serum (Beyotime) to block nonspecific binding; b) 200 μg / mL Nby-Aby fusion protein was added and incubated at 4°C overnight; c) Rabbit anti-HA monoclonal antibody (Proteintech, 1:200 dilution) was added and incubated at 37°C for 2 hours; d) HRP-labeled goat anti-rabbit secondary antibody (ZSGB-Bio) was incubated for 30 minutes, DAB color was developed (Zsbio), and hematoxylin was used for counterstaining.

[0055] (3) Pathological scoring and data analysis: HER2 scoring (0 / 1+ / 2+ / 3+) was performed according to the ASCO / CAP guidelines. The results were compared with those of traditional antibodies (Conv-Ab, Roche) and HER2-targeted nanoparticles (Nby-HFn and Aby-HFn) to test the effectiveness of Nby-Aby fusion protein in HER2 scoring in breast cancer tissue IHC detection. The results are shown in Figure 3 .

[0056] Figure 3 The experimental results showed that in some samples diagnosed as HER2 0 / 1+ / 2+ by traditional antibodies, the staining intensity was significantly enhanced when the Nby-Aby fusion protein was used for IHC staining. Statistical analysis showed that the results were statistically significant. This indicates that the Nby-Aby fusion protein provided by the present invention can effectively avoid the steric hindrance of HER2 dimers by dual-targeting and binding to regions I and III of the HER2 extracellular domain, while being able to simultaneously recognize HER2 monomers and dimers in tissues.

[0057] Test Example 2

[0058] Detection sensitivity comparison experiment and tissue microarray (TMA chip) large sample verification

[0059] a) TMA construction and detection

[0060] A TMA chip containing 100 breast cancer tissues (Shanghai National Biochip Center) was used. The Nby-Aby fusion protein was first labeled with HRP (refer to the instructions, Huzhou Yingchuang Biotechnology). After labeling, the Nby-Aby-HRP fusion protein was obtained, which was then dialyzed and the protein concentration was determined.

[0061] TMA chips were dewaxed according to IHC standards and hydrated. Endogenous peroxidase was blocked with 3% H2O2 and nonspecific binding was blocked with 5% goat serum (Beyotime). 200 μg / mL Nby-Aby-HRP fusion protein was added and incubated overnight at 4°C. After washing, DAB staining (Zsbio) was performed, and the slides were counterstained with hematoxylin and mounted for microscopic examination.

[0062] b) Comparison with the original diagnostic results of TMA

[0063] After 100 valid samples were tested in TMA, detected and scored using Nby-Aby-HRP fusion protein, and compared with the ISH, HER2 score and other information provided by the original TMA, statistical analysis showed the following conclusions:

[0064] 1) In the combined analysis of HER2 immunohistochemistry scores and ISH results, the Nby-Aby method had a significantly higher recognition rate for HER2-positive and low-expressing samples than the traditional antibody diagnostic method (89% vs 64%, P < 0.001), as shown in Table 1. This suggests that the Nby-Aby method has advantages in improving detection sensitivity and ISH consistency. Spearman correlation analysis with Ki67 (>30%) and tumor size (≤2 cm) showed a positive correlation trend between the Nby-Aby score and both (r = 0.61, P < 0.01), suggesting its potential in predicting tumor aggressiveness and early diagnostic sensitivity.

[0065] For samples provided by TMA that were originally diagnosed as HER2 (0 / 1+) by traditional methods, Nby-Aby-HRP re-evaluation can improve the scores of some diagnostic results from 0 / 1+ to 2+ or 3+ (see Figure 4 AB); at the same time, the diagnosis of the sample originally diagnosed as 2+ was 2+ or 3+ (see Figure 4 C); samples originally diagnosed as 3+ can also be diagnosed as 3+ (see Figure 4 D); Among them, Nby-Aby fusion protein is used for HER2 immune tissue patient detection, and its principle diagram of improving diagnostic sensitivity and result scoring level is as follows Figure 4 As shown in E.

[0066] The Ki67 index represents the proliferation of cell nuclei. The higher the index, the more vigorous the proliferation of cancer cells. The smaller the detection volume, the higher the sensitivity of detection under the same conditions. The ISH result is the RNA expression in cancer tissue and is usually used as the final diagnosis of positive or negative. For example, ICH++ but ISH- is usually diagnosed as HER2 negative. On the other hand, ISH+ and ICH++ are diagnosed as HER2 positive. When the method used in the present invention is combined with the ISH method, the original diagnosis of 63% positive rate can be increased to 89%, indicating that it is more consistent with the ISH diagnosis result. Therefore, the positive rate is also improved.

[0067] Table 1. Comparison of HER2 expression levels assessed by Nby-Aby diagnosis and initial diagnosis combined with other tumor markers

[0068]

[0069]

[0070] The test results conducted in the above-mentioned tissue microarray (TMA) and clinical breast cancer samples showed that the Nby-Aby fusion protein can, to a certain extent, enhance the ability to recognize HER2 signals in samples that are judged as HER2 low expression or negative by traditional methods, thereby improving the sensitivity and positive detection rate of detection.

[0071] The Nby-Aby fusion protein provided by the present invention improves the IHC detection signal and repeatability performance by dual-targeting HER2 and specifically binding to the non-dimerization region of HER2 under some experimental conditions (the Nby-Aby protein is labeled with HRP (i.e.), directly used for IHC detection conditions and incubated with the fusion protein Nby-Aby, incubated with rabbit anti-HA antibody and HRP-goat anti-rabbit IgG, and developed with DAB. Conventional IHC staining and color development) provides a reliable tool for accurate diagnosis and targeted treatment screening of HER2 low-expressing breast cancer. The present invention provides a fusion protein that avoids the steric hindrance of HER2 dimers and dual-targets HER2. It is predicted that its molecular weight is relatively small (23kDa), which can effectively improve its permeability in tissues, and the HA tag is fully exposed. The signal amplification efficiency used in immunohistochemical detection is significantly better than that of HER2-targeted nanoparticles (such as Nby-HFn), so it has high applicability in clinical diagnosis.

[0072] The fusion protein provided by the present invention is formed by connecting a nano antibody (Nby) and an affibody (Aby) that recognizes HER2 through a flexible connecting peptide (G4S). Wherein, Nby (PDB:5MY6_B) targets the extracellular domain I region of HER2, and Aby (GenBank:AEN27854.1) targets the extracellular domain III region. The fusion protein improves the possibility of binding to the HER2 molecule by simultaneously recognizing two spatially separated domains of HER2. In addition, since the sites to which Nby and Aby bind are different from the key domains (ECD II regions) related to the formation of HER2 dimers, the fusion protein is more accessible to the target antigen in tissue sections, thereby weakening the effect of the steric hindrance caused by HER2 dimers on antibody binding to a certain extent. Compared to traditional single-site antibodies, the fusion protein shows higher signal intensity in immunohistochemical detection, especially in breast cancer tissues where some HER2 is lowly expressed or determined to be negative by traditional methods, and its staining intensity and consistency are improved. The research results show that this fusion protein can provide an auxiliary tool for accurately evaluating HER2 expression levels in breast cancer patients, especially in cases involving low or borderline expression. It has potential clinical application value, helps to improve the screening sensitivity of relevant patients, and provides more accurate detection support for subsequent HER2 targeted therapy (such as T-DXd).

[0073] The present invention is not limited to the above-mentioned specific implementation methods. Various changes made by ordinary technicians in this field based on the above-mentioned conception without creative work are all within the scope of protection of this invention.

Claims

1. A fusion protein that avoids steric hindrance of HER2 dimers and dual-targets HER2, characterized in that: The fusion protein is formed by fusing Nby and Aby via a flexible connecting peptide G4S. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO:

2. Nby targets the extracellular region I of HER2, and its PBD database number is 5MY6_B; Aby targets the extracellular region III of HER2, and its GenBank number is AEN27854.

1.

2. The fusion protein according to claim 1 that avoids steric hindrance of HER2 dimers and dual-targets HER2, characterized in that: The binding regions of Nby and Aby are independent of the HER2 dimerization key region.

3. The fusion protein according to claim 1 that avoids steric hindrance of HER2 dimers and dual-targets HER2, characterized in that: The N-terminus of the fusion protein contains an HA tag.

4. The fusion protein according to claim 1 that avoids steric hindrance of HER2 dimers and dual-targets HER2, characterized in that: The amino acid sequence of the flexible connecting peptide G4S is Gly-Gly-Gly-Gly-Ser.

5. A method for preparing a fusion protein that avoids steric hindrance of HER2 dimers and dual-targets HER2 according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Synthesize the gene sequences of Nby and Aby respectively, and construct the fusion gene by introducing the flexible connecting peptide G4S; S2. Clone the fusion gene into the pET21a(+) expression vector and transform it into the E. coli BL21(DE3) host strain; S3. IPTG was used to induce host bacteria expression, and the fusion protein was obtained by Ni-NTA chromatography purification.

6. The preparation method according to claim 5, characterized in that The host bacteria induction conditions are: 37° C., 200 rpm shaking culture for 6 h, and a final IPTG concentration of 1 mM; the Ni-NTA chromatography purification uses 50-100 mM imidazole elution and PBS with a pH of 7.4 as the dialysate.

7. Use of the fusion protein according to any one of claims 1 to 4 in HER2 immunohistochemical detection.

8. The use according to claim 7, characterized in that The method for detecting HER2 by immunohistochemistry using fusion protein includes the following steps: A1. Tissue sample processing: Breast cancer tissue sections were dewaxed and antigen retrieval treated. Antigen retrieval conditions were: pH 6.0 sodium citrate buffer, incubation at 95°C for 20 min. A2. Sections were blocked with 3% H2O2 to block endogenous peroxidase and 5% goat serum to block nonspecific binding, and then incubated with 200 μg / mL Nby-Aby fusion protein at 4°C overnight or 37°C for 0.5-2 h. A3. Add rabbit anti-HA monoclonal antibody (Proteintech, 1:200 dilution) and incubate at 37°C for 2 h. Incubate with HRP-conjugated goat anti-rabbit secondary antibody (ZSGB-Bio) for 30 min. Develop with DAB and counterstain with hematoxylin. A3. After DAB staining, perform HER2 scoring according to the guidelines.

9. The use according to claim 7, characterized in that The method for detecting HER2 by immunohistochemistry using fusion protein includes the following steps: B1. Label the Nby-Aby fusion protein with HRP to obtain the Nby-Aby-HRP fusion protein, which is then dialyzed to determine the protein concentration; B2. The TMA chip containing breast cancer tissue was dewaxed and hydrated, and then blocked with 3% H2O2 to block endogenous peroxidase and 5% goat serum (Beyotime) to block non-specific binding. Finally, it was incubated with Nby-Aby-HRP fusion protein at a working concentration of 200 μg / mL at 4°C overnight, washed, developed with DAB, counterstained with hematoxylin, and mounted for microscopic examination.

10. A HER2 immunohistochemistry detection kit, characterized in that: The method comprises the fusion protein according to any one of claims 1 to 4, a DAB color developing solution and an HA tag antibody.

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