Truncated Gal d 1 allergen protein, nucleic acid molecule and application of truncated Gal d 1 allergen protein and nucleic acid molecule

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a truncated Gal d 1 allergen protein, a nucleic acid molecule and application of the truncated Gal d 1 allergen protein. The amino acid sequence of the truncated Gal d 1 allergen protein is as shown in SEQ. ID. NO.1. The invention also discloses a preparation method of the truncated Gal d 1 allergen protein. The truncated Gal d 1 allergen protein is obtained by modifying Gal d 1 by reasonably designing a truncated site, and an Elisa experiment shows that compared with commercial Gal d 1, the truncated Gal d 1 allergen protein has higher binding activity with IgE, so that the truncated Gal d 1 allergen protein has better biological activity and can be used for precise diagnosis of allergy and development of anti-allergic vaccines.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a truncated Gal d 1 allergen protein, nucleic acid molecule and its application. Background Technology

[0002] Gal d 1 (Gallus domesticus allergen 1) is a major allergenic protein in egg white, scientifically known as ovomucoid. Derived from domestic chicken (Gallus domesticus), it is also found in the egg whites of turkey and other poultry, accounting for 11% of the total protein in egg white. As a glycoprotein protease inhibitor with a molecular weight of approximately 28,000, Gal d 1 has an isoelectric point of 4.0 and exhibits significant thermostability, retaining its immunogenicity even in cooked egg white, making it a crucial trigger for egg allergy. Gal d 1 contains three domains, with the third domain showing particularly strong IgG and IgE binding activity. Its antigenic epitopes possess both linear and conformational properties, explaining its continued allergenicity in processed foods. Listed as one of the six egg allergens (Gal d 1 to Gal d 6), its specific IgE level is a key diagnostic indicator for predicting allergic reactions to heated eggs. Gal d 1 plays a significant role in the diagnosis and pathological mechanism research of egg allergy.

[0003] Truncation engineering, a common method for enhancing protein expression and activity, has received increasing attention in recent years. By removing or splicing portions of a protein sequence, truncation engineering can not only increase expression levels but also improve protein folding and stability.

[0004] Therefore, the present invention aims to truncate and modify the Gal d 1 allergen protein in order to obtain a truncated protein with higher biological activity. Summary of the Invention

[0005] This invention aims to address at least one of the problems existing in the prior art. To this end, this invention proposes a truncated Gald1 allergen protein, a nucleic acid molecule, and its applications. This invention modifies Gald1 by rationally designing truncation sites to obtain a truncated Gald1 allergen protein. ELISA experiments show that compared to commercial Gald1, the truncated Gald1 allergen protein has higher IgE binding activity, indicating that it has better biological activity and can be used for the precise diagnosis of allergies and the development of anti-allergy vaccines.

[0006] This invention provides a truncated Gal d 1 allergen protein, the amino acid sequence of which is shown in SEQ.ID.NO.1.

[0007] This invention modifies the Gal d 1 allergen protein (its amino acid sequence is shown in SEQ.ID.NO.2) by truncating the IgE binding site region. By truncating amino acids from position 1 (methionine, Met) to position 24 (glycine, Gly), a truncated Gal d 1 allergen protein with enhanced biological activity is obtained. ELISA experiments have confirmed that the truncated Gal d 1 allergen protein possesses higher IgE binding activity.

[0008] The present invention also provides a nucleic acid molecule for expressing the above-mentioned truncated Gal d 1 allergen protein.

[0009] Preferably, the nucleic acid molecule has undergone codon optimization.

[0010] Recombinant protein expression in *Escherichia coli* is a core technology in the field of biotechnology, widely used due to its high efficiency, economy, and ease of operation. *E. coli* offers advantages such as rapid growth, high protein yield (up to 50% of total protein), low culture cost, and mature genetic manipulation, making it suitable for producing therapeutic proteins (such as insulin), vaccines, and industrial enzymes. This invention optimizes the gene sequence of a truncated Gal d 1 allergen protein to match it with the tRNA library of *E. coli* strains, effectively increasing protein expression levels. Codon optimization involves adjusting codon usage patterns, replacing rare codons in the gene sequence with common, high-frequency codons in the host cell, based on the host cell's codon usage preferences. This makes the gene more adaptable to the host cell, improving translation efficiency and thus increasing protein expression levels.

[0011] The present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0012] The present invention also provides a recombinant bacterium containing the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector.

[0013] This invention also provides the application of the above-mentioned truncated Gal d 1 allergen protein in the preparation of allergen diagnostic kits.

[0014] The present invention also provides the application of the above-mentioned truncated Gal d 1 allergen protein in the preparation of anti-allergy vaccines.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs truncation modification technology to obtain a novel truncated Gal d 1 allergen protein through the rational design of truncation sites. ELISA experiments show that its IgE binding activity is 0.3377, exhibiting higher biological activity than the Gal d 1 protein, providing new insights for the precise diagnosis of allergies and the development of anti-allergy vaccines. Attached Figure Description

[0016] Figure 1 The results are obtained by SDS-PAGE analysis of the bacterial cell lysate sample after 6 hours of induction in Example 1.

[0017] Figure 2 The results are SDS-PAGE analysis of various samples in Example 1. Lane 1 is the supernatant sample after centrifugation of the bacterial cell lysate; Lane 2 is the flow-through liquid of the supernatant after centrifugation of the bacterial cell lysate bound to the nickel column; Lane 3 is the target protein eluted from the nickel column using 20 mM Tris-HCl buffer (containing 250 mM NaCl, 250 mM imidazole, pH 8.0).

[0018] Figure 3 The results are from the Western Blot analysis in Example 1; lane 1 contains purified Gal d 1-truncated protein elution buffer (20 μL loading volume), and the primary antibody is incubated with serum from an egg allergy patient (Gal d 1 IgE = 20.7 IU / L, diluted 1:4 with PBST solution).

[0019] Figure 4 The results of the enzyme-linked immunosorbent assay (ELISA) were used to compare the IgE binding activity of the truncated Gal d 1 allergen protein prepared in Example 1 with that of the commercial Gal d 1 full-length protein. Detailed Implementation

[0020] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0021] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0022] Gal d 1 can be obtained by searching for the sequence number P01005 on the UniPort website. Its amino acid sequence is shown in SEQ.ID.NO.2.

[0023] The nucleotide sequence of Gal d 1 after codon optimization for E. coli cells is shown in SEQ.ID.NO.3.

[0024] Example 1 This embodiment targets the Gal d1 allergen protein. Codon optimization was performed using E. coli cells provided by Aiji Biotechnology Co., Ltd., and the protein was truncated before its IgE binding site region. The amino acids from methionine (Met) to glycine (Gly) at position 1 were truncated, and the truncated gene was obtained using PCR amplification technology. This gene was then constructed into the pET-47b vector and expressed in the BL21(DE3) E. coli strain. Finally, a highly efficient, stable, and biologically active truncated Gal d1 allergen protein was screened.

[0025] The experimental steps are as follows: Using pET-47b plasmid as a template, primers were used for amplification to obtain the linear plasmid pET-47b-6his, which contains a 6his fusion tag sequence. Using a ready-to-use seamless cloning kit from Sangon Biotech, the obtained linear plasmid pET-47b-6his was seamlessly ligated with a truncated gene (Gal d 1-truncation) obtained by PCR amplification. The ligation product was transformed into T10 competent cells and plated on LB agar plates containing 50 ng / μL kanamycin, and incubated overnight at 37°C. Single colonies were grown for colony PCR verification. Plasmids containing the target band were extracted after culturing and sent to a sequencing company for sequence verification. The correctly sequenced plasmid pET-47b-6his-Gald 1-truncation was retained for later use, with the His tag located at the N-terminus of the Gal d 1 sequence.

[0026] Using pET-47b plasmid as a template, the following PCR amplification primers were designed to obtain the linear plasmid pET-47b-6his.

[0027] Forward: 5'-GCTCTGGTGCCACGCGTAG-3' (SEQ.ID.NO.4); Reverse: 5'-GCGGAGTGATGGTGGTGGTGATGTGCCATATGTATATCT-3' (SEQ.ID.NO.5); Using the full-length gene after codon optimization of Gal d1 as a template, the following PCR amplification primers were designed to amplify the Gal d1-truncated fragment.

[0028] Forward: 5'-ACCACCACCATCACTCCGCGGCAGAAGTTGACTGTTCCAGGTTTC-3' (SEQ.ID.NO.6); Reverse: 5'-CTACCGCGTGGCACCAGAGCttaGCACTTTCCAAAGTGGGATAAG-3' (SEQ.ID.NO.7); The amino acid sequence of the protein expressed by the Gal d 1-truncation fragment is: AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC (SEQ ID NO.1).

[0029] (2) The correctly sequenced plasmid pET-47b-6his-Gal d 1- was truncated and transformed into Escherichia coli (BL21(DE3)). The transformed bacteria were plated on LB agar plates containing 50 ng / μL kanamycin and incubated overnight at 37°C to screen for positive colonies. Positive transformants were verified by PCR. The target strain was cultured in LB agar at 37°C and 220 rpm on a shaker until the OD600 reached 0.8. Subsequently, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM to induce protein expression, and the culture was incubated at 25°C with shaking for about 6 hours.

[0030] (3) After expression, the bacterial cells were collected by centrifugation and resuspended in a buffer solution with the following formula: 20 mM Tris-HCl, 250 mM NaCl, 5 mM imidazole, pH 8.0. The cells were then broken up and the proteins were released by high-pressure cell disruption. The soluble protein supernatant was obtained by centrifugation and purified.

[0031] (4) The soluble protein supernatant was thoroughly mixed with the nickel column packing material. After loading the column, the flow-through was removed, and non-specifically bound proteins were washed away with a buffer solution containing 20 mM Tris-HCl, 250 mM NaCl, 5 mM imidazole, and pH 8.0. Then, the buffer solution containing 20 mM Tris-HCl, 250 mM NaCl, 250 mM imidazole, and pH 8.0 was used for elution to obtain the target protein - truncated Gal d 1 allergen protein.

[0032] Finally, the samples generated in each step of the purification process were subjected to SDS-PAGE and Western Blot analysis to verify protein expression and purity.

[0033] SDS-PAGE test results are as follows: Figure 1-2 As shown, the bacterial cell lysate clearly contains the target band, with a target protein yield reaching 90 mg / L. After binding with the nickel column packing material, the target protein in the flow-through solution significantly decreased, indicating that the target protein was effectively bound by the nickel column. Simultaneously, after elution with a high-concentration imidazole buffer solution, the target protein was fully eluted, with a protein concentration exceeding 95%.

[0034] Western blotting results are as follows: Figure 3 As shown, the results indicate that a clear band appeared at 25 kDa in lane 1, suggesting that IgE in the patient's serum can specifically bind to the Gal d 1 truncated protein.

[0035] Example 2 Using patient serum containing Gal d 1-specific IgE (20.7 kU / L), the IgE binding activity of the truncated Gal d 1 allergen protein prepared in Example 1 (i.e., Gal d 1 truncated) and the commercial full-length Gal d 1 protein (i.e., Gal d 1 full-length) was compared by enzyme-linked immunosorbent assay (ELISA). The experimental procedure included: coating two Gal d 1 proteins (10 mg / L) onto 96-well plates and incubating overnight at 4°C; blocking with 5% BSA for 2 hours, adding patient serum diluted 1:4 (Gal d 1 IgE = 20.7 IU / L), incubating at 37°C for 2 hours, incubating with HRP-labeled anti-human IgE secondary antibody (1:5000) for 2 hours; incubating with TMB at 37°C for 0.5 hours for color development, and measuring the OD value at 450 nm.

[0036] Test results are as follows Figure 4 The results showed that the OD 450 value of the truncated Gal d 1 allergen protein was 0.33, while the OD 450 value of the commercial full-length Gal d 1 protein was 0.13, indicating that the truncated Gal d 1 allergen protein has higher IgE binding activity compared to the commercial full-length Gal d 1 protein. The blank control had an OD 450 < 0.1, confirming the specificity of the experiment.

[0037] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application.

Claims

1. A truncated Gal d 1 allergen protein, characterized in that, The amino acid sequence of the truncated Gal d 1 allergen protein is shown in SEQ.ID.NO.

1.

2. A nucleic acid molecule, characterized in that, Used to express the truncated Gal d 1 allergen protein as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecules have undergone codon optimization.

4. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 2 or 3.

5. A recombinant bacterium, characterized in that, It comprises the nucleic acid molecule of claim 2 or 3 or the recombinant expression vector of claim 4.

6. The use of the truncated Gal d 1 allergen protein according to claim 1 in the preparation of an allergen diagnostic kit.

7. The use of the truncated Gal d 1 allergen protein according to claim 1 in the preparation of an anti-allergy vaccine.

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