Glycosylation modification form-based GtaA antigen and application thereof in aspergillus fumigatus infection detection

Abstract

The invention relates to the technical field of biological detection, and discloses a GtaA antigen based on a glycosylation modification form and application of the GtaA antigen in aspergillus fumigatus infection detection. The GtaA antigen is exosome glycoprotein from aspergillus, and the amino acid sequence of the GtaA antigen is as shown in SEQ ID NO: 2. The GtaA antigen comprises GtaA which is subjected to full glycosylation, GtaA (N) which is subjected to deglycosylation modification, and GtaA (N-O) which is subjected to non-glycosylation modification. The invention establishes an immunological detection method based on different forms of GtaA antigens. Experiments show that glycosylation modification affects antigen diagnosis performance, but GtaA in glycosylation and non-glycosylation forms can be used as specific diagnosis antigens of aspergillus infection. The invention is widely applicable to rapid detection of aspergillus fungal infection, including immunological detection modes such as enzyme-linked immunosorbent assay, immunochromatography, immunoblotting, chemiluminescence and the like, and has important clinical application potential and popularization value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for preparing a specific antigen GtaA derived from Aspergillus fungi and its glycosylated and non-glycosylated modified forms, as well as its application in the detection of Aspergillus infection. Background Technology

[0002] Aspergillus infections can lead to various clinical types of aspergillosis, primarily including invasive pulmonary aspergillosis (IA), chronic pulmonary aspergillosis (CPA), and allergic bronchial aspergillosis (ABPA). In recent years, the incidence of IA has increased significantly due to the widespread use of antibiotics, the prevalence of immunosuppressive therapy, and the increasing number of immunocompromised patients. Diagnosis and treatment of Aspergillus infections are particularly challenging in areas with insufficient medical resources or inadequate fungal disease surveillance systems. Furthermore, climate change and environmental shifts exacerbate the risk of Aspergillus spore transmission. It is estimated that there are over 2 million cases of IA globally each year; without timely diagnosis and treatment, the mortality rate exceeds 95%; however, early diagnosis and anti-Aspergillus treatment can significantly increase patient survival to 80%.

[0003] The difficulty in diagnosing Aspergillus infection is a major reason for its high pathogenicity and mortality. Current clinical diagnostic methods for fungal infections all have limitations: 1) Traditional fungal culture cycles are long (usually 2–3 weeks), and some Aspergillus strains are difficult to culture successfully, easily delaying optimal treatment; 2) Histopathological examination usually relies on surgery or puncture to obtain samples, posing significant risks to immunocompromised or critically ill patients, and the sample may not necessarily cover the infection focus; 3) Nucleic acid testing such as PCR and metagenomic sequencing requires high sample quality, and the tough cell walls of Aspergillus make DNA extraction difficult; metagenomic sequencing also relies on expensive equipment and professional personnel, making it difficult to promote in resource-limited areas.

[0004] Currently, the most widely used serological detection methods for Aspergillus infection internationally mainly include β-1,3-glucan (BG) detection and galactomannan (GM) detection. BG lacks species specificity and cannot distinguish Aspergillus from other opportunistic pathogens. GM, as a component of the Aspergillus cell wall, has been developed into various commercial kits and approved by the US, Europe, and the FDA. However, the sensitivity of GM detection varies considerably (29%–100%), and cross-reactivity with non-Aspergillus fungi, bacteria, drugs, and food components leads to false positives. Furthermore, GM detection shows uneven performance among different types of IA patients; the sensitivity can reach over 70% in patients with neutropenia, but only about 40% in patients without neutropenia. These shortcomings severely limit the clinical application of GM as a single diagnostic indicator. Serological methods have broad application prospects due to their simplicity, speed, and suitability for commercialization. Developing novel, species-specific, and highly stable Aspergillus antigens is key to improving diagnostic sensitivity and specificity. Previous studies have suggested that proteins and polysaccharides secreted by Aspergillus fungi during their growth have potential diagnostic value. However, research on whether Aspergillus exoglycoproteins can serve as antigenic markers for infection diagnosis is still relatively lacking, especially the impact of glycosylation and non-glycosylation modifications on their antigenic properties has not been systematically evaluated. Summary of the Invention

[0005] This invention aims to provide the application of GtaA, an exoglycoprotein derived from Aspergillus fungi, and its glycosylated and non-glycosylated forms, in the detection of Aspergillus infection. GtaA is a glycosylated protein secreted by Aspergillus fumigatus during hyphal growth, containing glycosylated side chains such as galactomannan (GM). Deglycosylation treatment confirmed the glycosylation characteristics of GtaA; further, heterologous expression in Escherichia coli yielded a non-glycosylated GtaA (NO−), and its antigenicity was verified. The results indicate that glycosylated GtaA, de-glycosylated GtaA (N−), and non-glycosylated GtaA (NO−) can all serve as antigens for Aspergillus infection detection, laying the foundation for the development of novel serological detection methods.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a GtaA protein derived from Aspergillus fumigatus, the amino acid sequence of which is shown in SEQ. ID No.2.

[0008] To further clarify, the GtaA protein is an exoglycoprotein GtaA, and its amino acid sequence is shown in SEQ. ID No. 2.

[0009] To further clarify, the GtaA protein is an N-glycosylated modified GtaA protein, labeled GtaA(N-), which is obtained by digesting GtaA protein with PNGase F, and its amino acid sequence is shown in SEQ ID No. 2.

[0010] To further clarify, the GtaA protein is a glycosylated GtaA protein, labeled GtaA(NO-), which is a recombinant protein obtained through expression in Escherichia coli, and its amino acid sequence is shown in SEQ ID No. 2.

[0011] The present invention also provides a recombinant expression vector comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ. ID No. 2, wherein the nucleotide sequence is shown in SEQ. ID No. 3 or SEQ. ID No. 4.

[0012] The present invention also provides a recombinant strain comprising the recombinant expression vector described above.

[0013] The present invention also provides a method for preparing the GtaA protein described above, comprising overexpressing GtaA in fungi or Escherichia coli and then purifying it.

[0014] The present invention also provides an immunological detection method, comprising using the GtaA protein described above as an antigen, coating or coupling the antigen to a solid-phase carrier, and using antibodies in the test sample for detection.

[0015] To further clarify, the detection method mentioned is enzyme-linked immunosorbent assay (ELISA), immunochromatography, Western blotting, colloidal gold immunoassay, chemiluminescence immunoassay, or other immunological detection methods.

[0016] The present invention also provides an Aspergillus infection detection kit, comprising the GtaA protein described above as an antigen.

[0017] The present invention also provides the application of the GtaA protein described above, the method for preparing the GtaA protein described above, the immunological detection method described above, or the kit described above in the preparation of products for detecting Aspergillus infections.

[0018] The amino acid sequence of GtaA protein in Aspergillus fumigatus is shown in SEQ ID NO.1:

[0019] .

[0020] The antigen has a full length of 691 amino acids, with the N-terminal 19 amino acids forming the signal peptide sequence "MQLFPLGVLIAFLASLAGA". Therefore, the mature Aspergillus fumigatus-derived glycoprotein GtaA contains 674 amino acids, with a theoretical molecular weight of 74.2 kDa, and its amino acid sequence is shown in SEQ. ID NO.2.

[0021] MQVSTFSPARPPALPLAVKSPYLSTWLSAGSDGGNGGYLAGEWPTFWEGQINGWAGLIRVDGAVYTWMGMPGSTTANQTAYEYTSTKSIFTFHVGDAVEMKVTFLSPITPNDLRRQSLTFSYVHVAVSSIDGNSHDVQLYSDISAEWVSGDRTAVAEWEYGVTHDGVAYHKVHRQTQLHFSEVRDQAEWGNWYWATEHSDRMTHQSGADVAVRGAFASNGTLGDSEDSNYRAISTNWPVFGFAIDMGSVTSSSVETHFTIGLAQSEAIQYSSPDGIRAEPSLWTSYFDDELAALEFFHYDYSTANKLSSALDERIARDSVAAAGQDYLTITSLSARQAFAATQLCGTLQNPYLFMKEISSNGNMNTVDVIFPAHPVFLYTNPELLELLMKPHFEIQESGQYPNAYAMHDIGTHYPNATGHPEGNDEPMPLEECGNMIIMALAYALKSSNTDYLNEHYPLLEQWTSYLVDEAIYPANQISTDDFAGPLANQTNLALKGIIGIEAMATISKLTHHADAATNRSAIAHDYIDRWQVLGIAHEADPPHTTLSYGSNDSHGLLYNLYADRELGLNLVPQSVYDMQSHFYPTVQKKYGVPLDTRHQYTKGDWELFTAAVASTSTRDMFIELLANWINQTPTNRALTDLYDTVNGDYPGITFIARPVMGGAFALLLLEEGL。

[0022] The gene encoding the above GtaA protein (antigen), the full-length gene sequence is shown in SEQ. ID NO.3:

[0023]

[0024] The present invention cloned and isolated the gene encoding the GtaA protein by PCR, with a full-length DNA of 2495 bp.

[0025]

[0026] The present invention has the following beneficial effects:

[0027] This invention utilizes GM antibodies to screen for a novel antigen for Aspergillus infection detection, along with its de-glycosylated and non-glycosylated mutants. The newly identified Aspergillus fumigatus antigen GtaA and its deglycosylated form exhibit significant and multifaceted beneficial effects in the diagnosis of Aspergillus infection. Compared to existing diagnostic methods, this invention utilizes the GtaA protein as an antigen, achieving high sensitivity and specificity in diagnostic performance through immunological detection methods (such as ELISA). The sensitivity reaches 81.25%, specificity 95.65%, positive predictive value 97.5%, negative predictive value 71.88%, and the area under the curve (AUC) reaches 0.9515, demonstrating significantly superior diagnostic efficacy compared to its deglycosylated form. Particularly in non-immunely deficient patients, the diagnostic performance is further improved (AUC = 0.9897), effectively compensating for the insufficient sensitivity of existing GM detection methods in this population. Furthermore, the antigen preparation process of this invention is simple, allowing for large-scale production using recombinant expression systems (such as Aspergillus fumigatus or Escherichia coli), resulting in low cost and suitability for widespread application in areas with limited medical resources. Serological testing requires only a small sample, is convenient to operate, and reacts rapidly, completing the test within hours. This facilitates early diagnosis and timely intervention, significantly improving patient survival rates. In summary, this invention not only possesses excellent sensitivity and specificity but also boasts comprehensive advantages such as ease of operation, low cost, rapid detection, and ease of promotion. It provides a reliable and efficient new approach for the clinical diagnosis of Aspergillus infection, possessing significant clinical application value and social benefits. Attached Figure Description

[0028] Figure 1 This is a diagram showing the SDS-PAGE electrophoresis results of GtaA expression in Aspergillus fumigatus.

[0029] Figure 2 The image shows the SDS-PAGE electrophoresis results of the de-N-glycosylated GtaA(N−) protein.

[0030] Figure 3 The image shows the SDS-PAGE electrophoresis results of GtaA(NO−) expressed and purified in Escherichia coli.

[0031] Figure 4 The graph shows the sensitivity and specificity of GtaA, GtaA(N-), and GtaA(NO) antigens in the serum of patients with different immune states, along with the ROC curve analysis results. Detailed Implementation

[0032] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0033] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0034] The experimental materials and reagents used in the specific implementation methods include:

[0035] 1. Strains and vectors:

[0036] Aspergillus fumigatus Ku80pyrG- strains and their recombinant expression vectors (such as pPTRII-GtaA), Escherichia coli expression strains (such as BL21(DE3) pLysS) and their expression vectors (such as pET-28a).

[0037] 2. Culture medium:

[0038] - LB medium, used for E. coli growth and protein expression.

[0039] LB liquid medium (1 L): 10 g peptone, 5 g yeast extract, 10 g sodium chloride.

[0040] LB solid medium (1 L): 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar powder.

[0041] YG and YGU media were used for the growth of Aspergillus fumigatus and the expression of exoproteins.

[0042] YG medium (1 L): 5 g yeast extract, 1 mL 1000× trace element solution, 20 g glucose.

[0043] YGU: YG medium, with uridine and uracil added to a final concentration of 5 mM.

[0044] - CD medium, used for selective culture of strains.

[0045] CD medium (1 L): 6.0 g sodium nitrate, 0.52 g potassium chloride, 1.52 g potassium dihydrogen phosphate, 2 mL 1 M magnesium sulfate heptahydrate, 47.6 g sodium chloride, 10.0 g glucose, 1 mL 1000× trace element solution, adjust pH to 6.5 with potassium hydroxide, and then bring the volume to 1 L with deionized water.

[0046] 3. Common biochemical reagents and enzymes:

[0047] DNA endonucleases, seamless cloning reagents, protein extraction buffers, etc., can all be obtained from commercially available companies.

[0048] 4. Buffer solution:

[0049] - Standard buffer solutions for DNA extraction, protein extraction and purification.

[0050] Example 1: This example demonstrates the overexpression of GtaA protein in Aspergillus fumigatus.

[0051] DNA extraction from the strain: Inoculation with Aspergillus fumigatus Ku80 pyrG stored at -80℃ - Spores were transferred to YGU agar plates and incubated upside down at 37°C for approximately 48 hours. Spores were then harvested using 0.2% Tween 20. Inoculation was performed at 1×10⁶ cells / day. 6 One *Aspergillus fumigatus* spore was cultured in a 1.5 mL EP tube containing 1 mL of YG liquid medium at 37°C and 220 rpm for 24 h until a certain amount of hyphae grew. The tube was centrifuged at 12000 g for 10 min, and the supernatant was aspirated and discarded. 300 µL of DNA extraction buffer was added, followed by the addition of a 1 mm diameter steel bead. The mixture was ground for 1 min, repeated twice, and incubated at -20°C for 10 min to remove foam. An equal volume of phenol-chloroform was added, and the mixture was inverted and centrifuged at 12000 g for 10 min. Layering was observed; the upper layer was transferred to a new 1.5 mL EP tube, and an equal volume of isopropanol was added. The tube was incubated at -20°C for at least 1 h. The tube was centrifuged at 12000 g for 10 min, the supernatant was discarded, and the tube was rinsed twice with 70% ethanol by pipetting and aspirating. The tube was then air-dried, dissolved in 50 µL of ddH2O, and the DNA concentration was determined using Nano Drop 2000. *Aspergillus fumigatus* genomic DNA was obtained and used for downstream PCR and plasmid construction.

[0052] Obtaining Aspergillus fumigatus cDNA: Cultured Aspergillus fumigatus hyphae were frozen at -80°C until use. A small amount of liquid nitrogen was added to a pre-sterilized mortar, followed by the Aspergillus fumigatus hyphae. The mixture was ground while adding liquid nitrogen until the hyphae were ground into a white powder. Total RNA was extracted using the Trizol extraction kit from TransGen. cDNA was then generated by reverse transcription using the Novizan reverse transcription kit.

[0053] Construction of the pPTRII-GtaA recombinant plasmid: Using pKS529 plasmid as a template, PCR amplification of fragment 1 (see Table 1) was performed using GTAA-1-F and GTAA-1-R primers. The PCR reaction parameters were: 98°C, 5 min, 1 cycle; 98°C, 10 sec, 62°C, 15 sec, 72°C, 25 sec, 30 cycles; 72°C, 10 min, 1 cycle. The amplified fragment 1 was 118 bp in size. Using Aspergillus fumigatus cDNA as a template, PCR amplification of fragment 2 was performed using GTAA-2-F and GTAA-2-R primers. The PCR reaction parameters were: 98°C, 5 min, 1 cycle; 98°C, 10 sec, 58°C, 30 sec, 72°C, 180 sec, 30 cycles; 72°C, 10 min, 1 cycle. The resulting fragment was 2050 bp in size. Fragment 1 and fragment 2 were ligated into the pCE-Zero plasmid using a seamless cloning method. The ligated recombinant plasmid was sent to Genewiz Biotechnology Co., Ltd. for sequencing and named pPTRII-GtaA. Using extracted Aspergillus fumigatus genomic DNA as a template,

[0054] Construction of pPTRII-GtaA Aspergillus fumigatus overexpression strain: Preparation of Aspergillus fumigatus Ku80pyrG - Protoplasts were transformed with pPTRII-GtaA and screened on MM + 1M sorbitol plates. The screening plates were incubated at 37°C for approximately 48 h, and the resulting transformants were picked to verify the selection marker and GtaA fragment, thus obtaining GtaA overexpressing strains.

[0055] Table 1 Primers required for constructing recombinant plasmid pPTRII-GtaA

[0056]

[0057] Example 2: This example describes the construction of the pET-28a-GtaA expression vector.

[0058] Using pET28 plasmid as a vector, the linearized pET28a plasmid was amplified using primer pairs pET28a-3-F and pET28a-3-R, yielding a target fragment of 5287 bp. Using Aspergillus fumigatus cDNA as a template, the GTAA cDNA sequence was amplified using primer pairs HGTAAH-F and HGTAAH-R (Table 2), yielding a fragment of 2041 bp. This fragment was then seamlessly ligated into the linearized pET28a vector.

[0059] Table 2 Primers required for constructing the pET-28a-GtaA expression vector

[0060]

[0061] Example 3: This example demonstrates the overexpression of GtaA protein in Aspergillus fumigatus and the expression and purification of GtaA(NO-) protein in Escherichia coli.

[0062] Expression of GtaA in Aspergillus fumigatus: Prepare fresh spores and inoculate 1×10⁶ cells onto CD medium plates containing 0.1 µg / mL PT. 8 Fresh spores were spread evenly with a coating stick and cultured under suitable conditions. The hyphae were collected, flash-frozen in liquid nitrogen, ground with liquid nitrogen, and then protein extraction buffer containing PMSF was added. The hyphae were removed by vacuum ultrafiltration to obtain a supernatant containing the target protein.

[0063] Expression of GtaA (NO-) in *E. coli*: The recombinant plasmid pET28A-GtaA expressing the *E. coli* protein was transformed into *E. coli* BL21 (DE3) plysS competent cells. The cells were inoculated at a 1:100 ratio into fresh LB broth containing the appropriate antibiotic and cultured under suitable conditions until OD600. 600 nm The concentration was set between 0.6 and 0.8, and then sterile self-induction medium was added to induce expression.

[0064] Protein purification: Equilibrate the packing material. Pack the chromatography column with 400 µL of packing material, wash with ddH2O first, then wash with protein extraction buffer until equilibration. Add filtered, sterile total protein and incubate on a shaker at 4°C for 1 h. Wash with 200 mL of ice-cold protein buffer, and elute the target protein with an imidazole gradient of 20 mM–250 mM. Verify the purified protein with 12% SDS-PAGE.

[0065] Example 4: This example is GtaA deglycosylation.

[0066] Mix 1 µL of 5% SDS and 1 µL of 1 M DTT with 12 µL of the target glycoprotein (50 µg) until homogeneous, for a total volume of 14 µL;

[0067] Denature the sample by boiling at 100°C for 10 min, then cool to room temperature for 5 min; add 2 µL of 0.5 M Na2HPO4 buffer (pH 7.5).

[0068] Add 2 µL of 10% NP-40 and 2 µL of deionized water, for a total volume of 20 µL. Add 1–2 µL of PNGase F and incubate at 37°C for 1–3 h to obtain the de-N-glycosylated GtaA protein (denoted as GtaA(N-)). Replace with a buffer solution to obtain purified GtaA(N-) protein for downstream immunological experiments or antigen analysis.

[0069] Example 5: This example demonstrates the application of GtaA, GtaA(N-), and GtaA(NO-) in the detection of serum antibodies in patients.

[0070] The 71 serum samples used for evaluating diagnostic methods for Aspergillus (Table 3) included 23 patients clinically diagnosed with Aspergillus infection, meeting one of the host factors, clinical criteria, and microbiological criteria; 25 patients tentatively diagnosed with Aspergillus infection, meeting one of the clinical criteria and one of the microbiological criteria; and 25 patients diagnosed with no evidence of Aspergillus infection. Two serum samples were used to establish the indirect ELISA method. One patient had a GM value of 5.49, lung shadows, and was a critically ill respiratory patient, initially diagnosed as positive for Aspergillus infection.

[0071] Table 3 Patient baseline

[0072]

[0073] Purified GtaA, GtaA(N-), and GtaA(NO-) proteins were used as coating antigens. Key parameters such as coating concentration, blocking buffer, serum dilution ratio and incubation conditions, secondary antibody dilution ratio and incubation time, and chromogenic substrate reaction time were optimized. A reproducible and stable indirect ELISA method was established to detect antibody reactions against GtaA protein and its glycosylated modified forms in serum (Table 4).

[0074] Table 4 Optimal ELISA reaction conditions for three Aspergillus fumigatus antigens

[0075]

[0076] Indirect ELISA detection method for GtaA: The optimal ELISA detection method established in the table above was followed. 100 µL of coated and purified GtaA, GtaA(N-), or GtaA(NO-) was added to each well of the ELISA plate and incubated overnight at 4°C. Wash 5 times with PBST for 3 min each time; add 200 µL of blocking buffer and block at 37°C for 1 h. Wash 5 times with PBST for 3 min each time; then add 100 µL of serum diluted to a certain ratio to each well and incubate at 37°C for 1 h. Wash 5 times with PBST for 3 min each time; add 100 µL of a diluted secondary antibody to each well and incubate at 37°C for 1 h. Wash 5 times with PBST for 3 min each time; mix the TMB chromogenic solution and add 100 µL to each well, incubate at room temperature in the dark; stop the reaction by adding 50 µL of 2 mol / L H2SO4; measure OD. 450 nm Absorbance value.

[0077] Precision of indirect ELISA: One sample was tested 20 times in parallel. The intra-assay coefficient of variation (CIG) for GtaA antigen was 6.15%, for GtaA(N-) antigen it was 6.01%, and for GTAA antigen it was 6.39%, all within 10%, indicating good precision. One sample was tested continuously for 20 days. The inter-assay CIG for GtaA(NO-) antigen was 11.80%, for GTAA-O it was 7.03%, and for GTAA antigen it was 7.84%, all within 15%, indicating good stability (Table 5).

[0078] Table 5. Intra-batch and inter-batch variability coefficients of three Aspergillus fumigatus antigens

[0079]

[0080] All three antigens can detect specific antibodies against Aspergillus infection in serum. GtaA antigen showed the highest sensitivity and specificity, followed by GtaA(N-), with GtaA(NO-) showing slightly lower sensitivity. Both false positive and false negative rates were within acceptable ranges, indicating the reliability of the antigens in clinical serological testing. Receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) of the antigens was largest for GtaA antigen, followed by GtaA(N-), and smallest for GtaA(NO-) (Table 6). These results further demonstrate the excellent performance of the antigens and their glycosylated modifications in the detection of Aspergillus infection.

[0081] Table 6 Diagnostic characteristics of three Aspergillus fumigatus antigens by ELISA

[0082]

[0083] Evaluation of serum testing methods in immunocompromised and non-immunocompromised patients: Based on patient medical records, we identified 8 patients clinically diagnosed or suspected of having Aspergillus infection who exhibited immunosuppression, including patients with acute leukemia, cancer, *Gnaphalium marneffei* infection, and myelodysplastic syndrome. Similarly, 10 patients in the non-Aspergillus infection group also showed immunosuppression based on their medical records. After grouping serum samples according to immunosuppression, we re-tested them using indirect ELISA methods with three different glycosylated GTAA antigens.

[0084] The results are as follows Figure 4In the GtaA antigen detection method, the AUC values ​​for non-immunodeficient and immunodeficient patients were 0.9897 and 0.9050, respectively; in the GatA(-O) antigen detection method, the AUC values ​​for non-immunodeficient and immunodeficient patients were 0.9128 and 0.8500, respectively; and in the GtaA(NO-) antigen detection method, the AUC values ​​for non-immunodeficient and immunodeficient patients were 0.8359 and 0.6400, respectively. The ROC curves of the ELISA diagnostic methods for all three antigens showed that the area under the curve was larger in non-immunodeficient patients than in immunodeficient patients. This indicates that the detection performance of the three GtaA antigens is superior in non-immunodeficient patients compared to immunodeficient patients.

[0085] In summary, specific embodiments of the present invention successfully obtained GtaA protein and its variants GtaA(N-) and GtaA(NO-) from *Aspergillus fumigatus* through cloning, expression, purification, and deglycosylation. Using an indirect ELISA method, 71 clinical serum samples were tested, confirming that the GtaA antigen exhibits excellent performance in the detection of *Aspergillus* infection: sensitivity of 81.25%, specificity of 95.65%, AUC value of 0.9515, and good precision (intra-assay coefficient of variation ≤ 6.39%). Comparative studies show that the diagnostic efficacy of GtaA is significantly better than that of the deglycosylated form, highlighting the crucial role of glycosylation modification in antigen-antibody recognition. Furthermore, GtaA demonstrated high diagnostic accuracy in both immunodeficient and non-immunodeficient patients, especially in the non-immunodeficient population where the AUC approached 0.99. These results fully validate the reliability and practicality of GtaA as a novel antigen for detecting *Aspergillus* infection, laying a solid foundation for the development of rapid and accurate diagnostic kits and possessing broad clinical application prospects.

[0086] Although the present invention has been described in detail above, including general descriptions and specific embodiments, the technical solutions of the present invention can be appropriately modified or improved according to actual needs, and such modifications or improvements will be obvious to those skilled in the art. Therefore, any equivalent substitutions, adjustments, or optimizations made to the present invention without departing from the spirit and essence of the invention should be considered within the scope of protection claimed by the present invention.

Claims

1. A GtaA protein derived from Aspergillus fumigatus, characterized in that, Its amino acid sequence is shown in SEQ ID No.

2.

2. The GtaA protein according to claim 1, characterized in that, The GtaA protein is an exoglycoprotein GtaA, and its amino acid sequence is shown in SEQ ID No.

2.

3. The GtaA protein according to claim 1, characterized in that, The GtaA protein is an N-glycosylated modified GtaA protein, labeled GtaA(N-), which is obtained by digesting GtaA protein with PNGase F, and its amino acid sequence is shown in SEQ ID No.

2.

4. The GtaA protein according to claim 1, characterized in that, The GtaA protein is a glycosylated GtaA protein, labeled GtaA(NO-), which is a recombinant protein obtained by expression in Escherichia coli, and its amino acid sequence is shown in SEQ ID No.

2.

5. A recombinant expression vector, characterized in that, A nucleotide sequence comprising the amino acid sequence shown in SEQ ID No. 2, said nucleotide sequence being shown in SEQ ID No. 3 or SEQ ID No.

4.

6. A method for preparing the GtaA protein of claim 1, characterized in that, This includes overexpressing GtaA in fungi or Escherichia coli and then purifying it.

7. An immunological detection method, characterized in that, This includes using the GtaA protein of claim 1 as an antigen, coating or coupling the antigen to a solid-phase carrier, and using antibodies in the test sample for detection.

8. The detection method according to claim 6, characterized in that, The method is enzyme-linked immunosorbent assay (ELISA), immunochromatography, immunoblotting, colloidal gold immunoassay, chemiluminescence immunoassay, or other immunological detection methods.

9. A test kit, characterized in that, The kit contains the GtaA protein of claim 1 as an antigen.

10. The use of the GtaA protein of claim 1, the method for preparing the GtaA protein of claim 6, the immunological detection method of claim 7 or 8, or the kit of claim 9 in the preparation of products for the detection of Aspergillus infections.

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