Truncated tropomyosin, nucleic acid molecule and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to truncated tropomyosin, a nucleic acid molecule and application of the truncated tropomyosin. The truncated tropomyosin is based on an alpha-helical structure of tropomyosin, and is obtained by taking glutamic acid (E26) at the 26th site, glutamine (Q51) at the 51st site or glutamic acid (E73) at the 73rd site as a truncation site. By reasonably designing a truncation site and further replacing the secretion signal peptide, the expression quantity of the protein in a recombinant expression system is remarkably improved, the immunogenicity of the protein is effectively reserved, and the protein can be used for precise diagnosis of allergy and development of anti-allergic vaccines.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a truncated tropomyosin, a nucleic acid molecule and applications thereof. Background Art

[0002] Tropomyosin is a class of macromolecular motor proteins widely found in animal cells, primarily responsible for cell motility, muscle contraction, and many other essential cellular functions. Tropomyosin works together with actin, utilizing energy provided by ATP (adenosine triphosphate) to generate mechanical movement within the cell. It is the core driver of muscle fiber contraction and also plays a vital role in processes such as cell division, intracellular cargo transport, and tissue remodeling. During muscle contraction, tropomyosin binds to actin, generating a sliding action that shortens muscle fibers, thereby achieving muscle contraction and movement. This process is driven by energy provided by ATP hydrolysis. Tropomyosin binds to actin through its head region, generating force through a "sliding fiber" mechanism.

[0003] Tropomyosin 1 (Penaeus aztecus tropomyosin 1) is an allergen secreted by shrimp (Penaeus aztecus). It belongs to the tropomyosin family and is a class of allergenic proteins that can trigger IgE-mediated immune responses in humans. Pen a 1 is a core sensitizer in crustacean allergies and is particularly immunogenic in patients with seafood allergies. It can trigger mast cell degranulation, causing symptoms ranging from skin urticaria to anaphylactic shock, and cross-reacts with tropomyosins from invertebrates such as dust mites. Through in vitro expression of recombinant Pen a 1 protein, it can be used as a target for specific IgE detection in clinical diagnosis, accurately identifying individuals with seafood allergies.

[0004] Tropomyosin 10 (Dermatophagoides pteronyssinus), derived from the house dust mite (Dermatophagoides pteronyssinus), is a member of the tropomyosin family and is officially listed as a major dust mite allergen by the World Health Organization (WHO / IUIS). Der p 10 has a highly conserved α-helical structure and shares significant sequence homology with tropomyosins from invertebrates, including crustaceans (such as shrimp and crab), mollusks (such as shellfish), and insects (such as cockroaches), resulting in strong cross-reactivity. Its sensitization mechanism primarily involves binding to specific IgE in allergic patients, triggering mast cell degranulation and the release of inflammatory mediators such as histamine, leading to allergic reactions. Due to cross-sensitization, some individuals with dust mite allergies may also experience cross-sensitization to seafood, owing to the sequence similarity between Der p 10 and tropomyosins from crustaceans and mollusks. Furthermore, Der p 10 plays a key role in airway inflammation such as asthma and allergic rhinitis by promoting Th2 immune response.

[0005] Truncation engineering, a common method for improving protein expression and activity, has garnered increasing attention in recent years. By removing or splicing portions of a protein sequence, truncation engineering can not only boost expression but also improve protein folding and stability. Therefore, the present invention aims to perform truncation engineering on tropomyosin, aiming to obtain truncated tropomyosin with higher yields and retained biological activity. Summary of the Invention

[0006] The present invention aims to address at least one of the problems existing in the aforementioned prior art. To this end, the present invention provides a truncated tropomyosin, a nucleic acid molecule, and its use. By rationally designing the truncation site and further replacing the secretion signal peptide, its expression level in a recombinant expression system is significantly increased while its immunogenicity is effectively retained. It can be used for accurate diagnosis of allergies and development of anti-allergy vaccines.

[0007] The present invention provides a truncated tropomyosin. The truncated tropomyosin is based on the α-helical structure of tropomyosin and is obtained with glutamic acid at position 26 (E26), glutamine at position 51 (Q51) or glutamic acid at position 73 (E73) as a truncation site.

[0008] The present invention can increase protein expression through truncation design and retain key IgE binding sites. The IgE binding activity of the truncated tropomyosin has been confirmed by ELISA experiments, ensuring that the immunogenicity is equivalent to that of the full-length protein.

[0009] Preferably, the truncation site is glutamine 51 (Q51).

[0010] Preferably, the tropomyosin is Pen a 1 or Der p 10.

[0011] More preferably, when the tropomyosin is Pen a 1, the amino acid sequence of the truncated tropomyosin is as shown in SEQ ID NO. 1-3.

[0012] More preferably, when the tropomyosin is Der p 10, the amino acid sequence of the truncated tropomyosin is as shown in SEQ ID NO. 4-6.

[0013] The present invention also provides a nucleic acid molecule for expressing the truncated tropomyosin.

[0014] Preferably, the nucleic acid molecule is codon-optimized. The present invention performs codon optimization on the gene sequence of truncated tropomyosin to match it with the tRNA library of Pichia pastoris GS115, which can effectively increase protein expression. Codon optimization is achieved by adjusting the codon usage pattern, replacing rare codons in the gene sequence with common high-frequency codons in the host cell based on the codon usage preference of the host cell, making it more adaptable to the host cell and improving translation efficiency, thereby increasing protein expression levels.

[0015] The present invention also provides a recombinant expression vector comprising the above nucleic acid molecule.

[0016] Preferably, the recombinant expression vector further comprises a Bla g2 signal peptide gene. By introducing the Bla g2 signal peptide, the expression efficiency of truncated tropomyosin in Pichia pastoris can be significantly improved, and protein degradation can be effectively reduced.

[0017] The present invention also provides a recombinant bacterium comprising the above nucleic acid molecule or the above recombinant expression vector.

[0018] The present invention also provides the use of the truncated tropomyosin in preparing an allergen diagnostic kit.

[0019] The present invention also provides the use of the truncated tropomyosin in preparing anti-allergic vaccines.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts truncation transformation and codon optimization technology, by rationally designing truncation sites and replacing secretion signal peptides. By truncation transformation, the expression level of Pen a 1 protein is increased to 50 mg / L, and the Elisa experiment and IgE binding activity data are 0.6. By truncation transformation and replacing the secretion signal peptide, the expression level of Der p 10 protein is increased by 3 times, and the Elisa experiment result of binding activity with IgE is 0.33. The present invention breaks through the bottleneck of low efficiency of full-length sequence expression, difficulty in folding conformation heterogeneity and insufficient biological activity, and provides a kind of efficient, stable and biologically active truncated tropomyosing recombinant protein, which significantly improves its expression level and biological activity in the recombinant expression system, and provides new ideas for the accurate diagnosis of allergies and the development of anti-allergic vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a Western Blotting diagram of the expression results of Pen a 1 in Pichia pastoris; lane 1 is Pen a 1-full, lane 2 is Pen a 1-E26-cut, lane 3 is Pen a 1-Q51-cut, and lane 4 is Pen a 1-E73-cut.

[0022] Figure 2 for Figure 2 This is an SDS-PAGE image of the expression results of Der p 10 in Pichia pastoris after replacing the secretion signal peptide; lane 1 is Bla g2-sig-Der p 10-1; lane 2 is Bla g2-sig-Der p 10-2; lane 3 is Bos d6-sig-Der p 10; lane 4 is Can f1-sig-Der p 10; lane 5 is Can f1-sig-Der p 10; lane 6 is Can f6-sig-Der p 10-1; lane 7 is Can f6-sig-Der p 10-2; lane 8 is Equ c1-sig-Der p 10; lane 9 is Fel d2-sig-Der p 10-1; lane 10 is Fel d2-sig-Der p 10-2.

[0023] Figure 3Western Blotting diagram of the expression results of Der p 10 in Pichia pastoris; lane 1 is Der p 10-full expressed with the α-factor secretion signal peptide, lane 2 is Der p 10-Q51-cut expressed with the α-factor secretion signal peptide, lane 3 is Der p 10-E73-cut expressed with the α-factor secretion signal peptide, and lane 4 is Der p 10-Q51-cut expressed after replacing the Blag2 secretion signal peptide.

[0024] Figure 4 for Figure 3 Lane 1 in the middle is Der p 10-full expressing the α-factor secretion signal peptide. Since the overall exposure effect was not ideal and no bands could be seen, it was re-exposed and developed separately.

[0025] Figure 5 This is the result of Elisa test on target protein in serum with IgE=99. DETAILED DESCRIPTION

[0026] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0027] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0028] Pen a 1-full can be obtained through the UniPort official website by querying the sequence number Q3Y8M6 to obtain detailed information about the protein.

[0029] The nucleotide sequence of Pen a 1-full after codon optimization suitable for yeast cells is shown in SEQ.ID.NO.7.

[0030] Der p 10-full protein details can be obtained by searching the sequence number O18416 on the UniPort official website.

[0031] The nucleotide sequence of Der p 10-full after codon optimization suitable for yeast cells is shown in SEQ.ID.NO.8.

[0032] Example 1

[0033] This example targets the α-helical conformation of Pen a 1 and Der p 10 tropomyosin (Pen a 1 and Der p 10 tropomyosin proteins provided by Aiki Biotech Co., Ltd.). By truncating the proteins before their IgE binding site, we selected glutamic acid 26 (E26), glutamine 51 (Q51), and glutamic acid 73 (E73) as truncation sites. PCR amplification was used to obtain the truncated genes, which were then constructed into the pPIC9K vector (containing the α-factor signal peptide sequence) and expressed in the GS115 Pichia pastoris strain. The truncation site that ultimately resulted in efficient expression was identified as glutamine 51 (Q51). The experimental steps are as follows: (1) Using the pPIC9K plasmid as a template, primers were used for amplification to obtain the linearized plasmid pPIC9K-α-factor, which contains the α-factor signal peptide sequence. Using the ready-to-use seamless cloning kit from Sangon Biotech Co., Ltd., the obtained linearized plasmid was seamlessly linked with the truncated gene obtained by PCR amplification technology. The linked product was transformed into T10 competent cells, plated on a plate containing ampicillin, and cultured at 37°C overnight. After the growth of monoclonal colonies, colony PCR verification was performed. After culturing the single colony containing the target band, the plasmid was extracted and sent to a sequencing company for sequence verification. The correctly sequenced plasmids pPIC9K-α-factor-Pen a 1-E26-cut, pPIC9K-α-factor-Pen a 1-Q51-cut, pPIC9K-α-factor-Pen a 1-E73-cut, pPIC9K-α-factor-Der p 10-E26-cut, pPIC9K-α-factor-Der p 10-Q51-cut, and pPIC9K-α-factor-Der p 10-E73-cut were kept for later use.

[0034] Among them, using the pPIC9K plasmid as a template, the following PCR amplification primers were designed to obtain the pPIC9K-α-factor linear plasmid.

[0035] Forward: 5'-CACCACCATCACCACCATTAAGCGGCCGCGAATTAATTCG-3' (SEQ.ID.NO.9); Reverse: 5'-AGCTTCAGCCTCTCTTTTCTCGAGA-3' (SEQ.ID.NO.10); Using the codon-optimized full-length gene of Pen a 1 as a template, the following PCR amplification primers were designed to amplify the Pen a 1-E26-cut fragment.

[0036] Forward: 5'-AGAAAGAGAGGCTGAAGCTCAGCAGAACAAGGAGGCCAACAAC-3' (SEQ.ID.NO.11); Reverse: 5'-TAATGGTGGTGATGGTGGTGGTAGCCAGACAGTTCGCTGAAAGTC-3' (SEQ.ID.NO.12); Pen a The amino acid sequence of the protein expressed by the 1-E26-cut fragment is: QQNKEANNRAEKSEEEVHNLQKRMQQLENDLDQVQESLLKANIQLVEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERMRKVLENRSLSDEERMDALE NQLKEARFLAEEADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLKAAEARAEFAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY (SEQ ID NO.1).

[0037] Using the codon-optimized full-length gene of Pen a 1 as a template, the following PCR amplification primers were designed to amplify the Pen a 1-Q51-cut fragment.

[0038] Forward: 5'-AGAAAGAGAGGCTGAAGCTCAATTGGAGAATGACTTGGATCAGG-3' (SEQ.ID.NO.13); Reverse: 5'-TAATGGTGGTGATGGTGGTGATAACCAGACAATTCAGAAAATGTC-3' (SEQ.ID.NO.14); The amino acid sequence of the protein expressed by the Pen a 1-Q51-cut fragment is: QLENDLDQVQESLLKANIQLVEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERMRKVLENRSLSDEERMDALENQLKEARFLAEEADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLKAAEARAEFAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY (SEQ ID NO. 2); Using the codon-optimized full-length gene of Pen a 1 as a template, the following PCR amplification primers were designed to amplify the Pen a 1-E73-cut fragment.

[0039] Forward: 5'-AGAAAGAGAGGCTGAAGCTAAGGACAAAGCACTGTCTAATGCCG-3' (SEQ.ID.NO.15); Reverse: 5'-TAATGGTGGTGATGGTGGTGATAACCAGACAATTCAGAAAATGTC-3' (SEQ.ID.NO.16); The amino acid sequence of the protein expressed by the Pen a 1-E73-cut fragment is: KDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERMRKVLENRSLSDEERMDALENQLKEARFLAEEADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLKAAEARAEFAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY (SEQ ID NO.3).

[0040] Using the codon-optimized full-length gene of Der p 10 as a template, the following PCR amplification primers were designed to amplify the Der p 10-E26-cut fragment.

[0041] Forward: 5'-AGAAAAGAGAGCTGAAGCTCAGAAAGCTAGAGATGCCAACTTG-3' (SEQ.ID.NO.17); Reverse: 5'-TAATGGTGGTGATGGTGGTGGTAGCCAGACAGTTCGCTGAAAGTC-3' (SEQ.ID.NO.18); The amino acid sequence of the protein expressed by the Der p 10-E26-cut fragment is: QKARDANLRAEKSEEEVRALQKKIQQIENELDQVQEQLSAANTKLEEKEKALQTAEGDVAALNRRIQLIEEDLERSEERLKIATAKLEEASQSADESERMRKMLEHRSITDEERMEGLENQLKEARMMAEDADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKAQQREEAHEQQIRIMTTKLKEAEARAEFAERSVQKLQKEVGRLEDELVHEKEKYKSISDELDQTFAELTGY (SEQ ID NO.4).

[0042] Using the codon-optimized full-length gene of Der p 10 as a template, the following PCR amplification primers were designed to amplify the Der p 10-Q51-cut fragment.

[0043] Forward: 5'-AGAAAGAGAGGCTGAAGCTCAAATAGAGAACGAATTGGATCAGGTTC-3' (SEQ.ID.NO.19); Reverse: 5'-TAATGGTGGTGATGGTGGTGGTAGCCAGACAGTTCGCTGAAAGTC-3' (SEQ.ID.NO.20); The amino acid sequence of the protein expressed by the Der p 10-Q51-cut fragment is: QIENELDQVQEQLSAANTKLEEKEKALQTAEGDVAALNRRIQLIEEDLERSEERLKIATAKLEEASQSADESERMRKMLEHRSITDEERMEGLENQLKEARMMAEDADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKAQQREEAHEQQIRIMTTKLKEAEARAEFAERSVQKLQKEVGRLEDELVHEKEKYKSISDELDQTFAELTGY (SEQ ID NO. 5).

[0044] Using the codon-optimized full-length gene of Der p 10 as a template, the following PCR amplification primers were designed to amplify the Der p 10-E73-cut fragment.

[0045] Forward: 5'-AGAAAAGAGAGCTGAAGCTAAGGAAAAGGCCTTACAGACCGCAG-3' (SEQ.ID.NO.21); Reverse: 5'-TAATGGTGGTGATGGTGGTGGTAGCCAGACAGTTCGCTGAAAGTC-3' (SEQ.ID.NO.22); The amino acid sequence of the protein expressed by the Der p 10-E73-cut fragment is: KEKALQTAEGDVAALNRRIQLIEEDLERSEERLKIATAKLEEASQSADESERMRKMLEHRSITDEERMEGLENQLKEARMMAEDADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKAQQREEAHEQQIRIMTTKLKEAEARAEFAERSVQKLQKEVGRLEDELVHEKEKYKSISDELDQTFAELTGY (SEQ ID NO.6).

[0046] (2) The correctly sequenced plasmids pPIC9K-α-factor-Pen a 1-E26-cut, pPIC9K-α-factor-Pen a 1-Q51-cut, pPIC9K-α-factor-Pen a 1-E73-cut, pPIC9K-α-factor-Der p 10-E26-cut, pPIC9K-α-factor-Der p 10-Q51-cut, and pPIC9K-α-factor-Der p 10-E73-cut were transformed into Pichia pastoris (GS115 strain) and positive colonies were screened on MD plates. High-copy strains were screened on 6 mg / mL G418 plates. Positive transformants were verified by PCR. The target strains were cultured in BMGY medium at 30°C and shaken at 220 rpm. After 24 hours, the methanol induction medium BMMY was replaced and cultured. Methanol was supplemented every 24 hours to a final concentration of 1%. After 96 hours of induction culture, the bacteria were harvested and the expression of the target protein was detected. Take 30 μL of the fermentation broth of Pichia pastoris strain after 96 hours of fermentation for Western Blotting detection. The results are as follows: Figure 1 shown.

[0047] Depend on Figure 1 It can be seen that the full-length size of Pen a 1 is 36KDa, corresponding to the band indicated by the red arrow in lane 1. The comparison marker is consistent with the actual size. Through observation and comparison, it can be seen that the Pen a 1-Q51-cut band in lane 3 is the most obvious and deepest, and the protein expression level is increased to 50mg / L, indicating that truncation at position 51 glutamine (Q51) has the greatest advantage in increasing the production of the allergen component tropomyosin.

[0048] Example 2

[0049] (1) Using the pPIC9K-α-factor-Der p 10-full plasmid in Example 1 as a template, primers were used for amplification to obtain the linearized plasmid pPIC9K-Der p 10-full. This vector fragment does not have a signal peptide sequence. Then, using the ready-to-use seamless cloning kit of Sangon Biotech Co., Ltd., the obtained linearized plasmid was seamlessly cloned and linked with different signal peptide genes obtained by PCR amplification technology. The linked products were transformed into T10 competent cells, spread on plates with ampicillin, and cultured at 37°C overnight. After the growth of monoclonal colonies, colony PCR verification was performed. After culturing the single colony containing the target band, the plasmid was extracted and sent to a sequencing company to detect the correctness of the sequence. The correctly sequenced plasmids pPIC9K-Can f1 signal-Der p 10-full, pPIC9K-Can f6 signal-Der p 10-full, pPIC9K-Fel d2 signal-Der p10-full, pPIC9K-Bos 6 signal-Der p 10-full, pPIC9K-Equ c1 signal-Der p 10-full, and pPIC9K-Bla g2 signal-Der p 10-full were retained for future use.

[0050] Among them, using the pPIC9K-α-factor-Der p 10-full plasmid as a template, the following PCR amplification primers were designed to obtain the pPIC9K-Der p 10-full linear plasmid. The vector fragment contains the full-length sequence of Der p 10.

[0051] Forward: 5'-GAAGCCATTAAGAATAAGATGCAAGCC-3' (SEQ.ID.NO.23); Reverse: 5'-CGTTTGGATCCTTCGAATAATTAGTTG-3' (SEQ.ID.NO.24); The nucleotide sequences of Can f1 signal, Can f6 signal, Fel d2 signal, Bos 6 signal, Equ c1 signal, and Bla g2 signal are shown in SEQ.ID.NO.25-30.

[0052] (2) The correctly sequenced plasmids pPIC9K-Can f1 signal-Der p 10-full, pPIC9K-Can f6 signal-Der p 10-full, pPIC9K-Fel d2 signal-Der p 10-full, pPIC9K-Bos 6 signal-Der p 10-full, pPIC9K-Equ c1 signal-Der p 10-full, and pPIC9K-Bla g2 signal-Der p10-full were transformed into Pichia pastoris (GS115 strain) respectively. Positive colonies were screened on MD plates, and high-copy strains were screened on 6 mg / mL G418 plates. Positive transformants were verified by PCR. The target strains were cultured in BMGY medium at 30°C in a constant temperature shaker at 220 rpm. After 24 hours, the methanol induction medium BMMY was replaced and culture continued. Methanol was supplemented every 24 hours to a final concentration of 1%. After 96 hours of induction culture, the bacteria were harvested and the expression of the target protein was detected. 30 μL of the fermentation broth of the Pichia pastoris strain induced by 96 hours of fermentation was taken for SDS-PAGE detection. The results were as follows: Figure 2 shown.

[0053] Depend on Figure 2 As can be seen, Der p 10 is 36 kDa in size, corresponding to the band indicated by the red arrow in lane 1. The marker is consistent with its actual size, and the band in lane 1 is the most obvious and abundant, indicating that this Bla g2 secretion signal peptide is suitable for efficient expression of the allergen tropomyosin.

[0054] Example 3

[0055] Using the pPIC9K-Bla g2 signal-Der p 10-full plasmid as a template, primers were used for amplification to generate the linearized plasmid pPIC9K-Bla g2 signal. The linearized plasmid was seamlessly linked to Der p 10-Q51-cut using the Sangon Biotechnology Co., Ltd. ready-to-use seamless cloning kit. The ligation product was transformed into T10 competent cells, plated on plates supplemented with ampicillin, and incubated overnight at 37°C. Single colonies were generated and verified by colony PCR. Single colonies containing the target band were cultured and the plasmid was extracted and sent to a sequencing company for sequence verification. The correctly sequenced plasmid was retained as pPIC9K-Bla g2 signal-Der p 10-Q51-cut for future use.

[0056] Among them, using the pPIC9K-Bla g2 signal-Der p 10-full plasmid as a template, the following PCR amplification primers were designed to obtain the pPIC9K-Bla g2 signal linear plasmid, and the vector fragment contained the Bla g2 signal peptide sequence.

[0057] Forward: 5'-CACCACCATCACCACCATTAAGCGGCCGCGAATTAATTCG-3' (SEQ.ID.NO.31); Reverse: 5'-TGCATGTGTTATGGTAGCAACCGCAAAGAG-3' (SEQ.ID.NO.32); The correctly sequenced plasmid pPIC9K--Bla g2 signal-Der p 10-Q51-cut was transformed into Pichia pastoris (GS115 strain), and positive colonies were screened on MD plates, and high-copy strains were screened on 6 mg / mL G418 plates. PCR verified the positive transformants, and the target strain was cultured in BMGY medium at 30°C with a constant temperature shaker at 220 rpm. After 24 hours, the methanol induction medium BMMY was replaced and continued to be cultured. Methanol was supplemented with a final concentration of 1% every 24 hours. The induction culture was performed for 96 hours and the bacteria were harvested to detect the expression of the target protein. 30 μL of the fermentation broth of the Pichia pastoris strain induced by 96 hours of fermentation and the fermentation broth in Example 1 were taken for Western Blotting detection, and the results are shown as follows. Figure 3-4 shown.

[0058] Depend on Figure 3-4 As can be seen, the full-length Der p 10 is 36 kDa, corresponding to the band indicated by the red arrow in lane 1. Comparison with the marker confirms its actual size. Observation of the Der p 10-Q51-cut band in lane 2 shows a significant increase, indicating that truncation at position 51 (Gln51) is most beneficial for increasing the yield of the allergen component tropomyosin, raising protein expression to 15 mg / L. However, numerous minor bands were observed below the target band, suggesting that the target protein was degraded during the truncation. Comparison with Der p 10-Q51-cut in lane 4, expressed after replacing the Bla g2 secretion signal peptide, reveals clear bands without any other bands. The band is the most prominent and darkest, indicating that replacing the Bla g2 secretion signal peptide and simultaneously truncating at position 51 (Gln51) is most beneficial for increasing the yield of the allergen component tropomyosin and effectively addressing protein degradation.

[0059] Example 4

[0060] The target protein obtained in the above example was subjected to Elisa test under the condition of serum with IgE=99. The results are as follows: Figure 5 As shown (larger values ​​indicate stronger IgE binding activity). Observation and comparison revealed that tropomyosin binding activity was strongest after truncating at position 51 (Gln51), and that the tropomyosin binding activity was significantly enhanced 2-3 times after replacing the Bla g2 secretion signal peptide.

[0061] The embodiments of the present application are described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the purpose of the present application.

Claims

1. A truncated tropomyosin, characterized in that The truncated tropomyosin is based on the α-helical structure of tropomyosin and is obtained by using the 26th glutamic acid, the 51st glutamine or the 73rd glutamic acid as the truncation site.

2. The truncated tropomyosin according to claim 1, characterized in that The truncation site is the 51st glutamine.

3. The truncated tropomyosin according to claim 1, wherein The tropomyosin is Pen a 1 or Der p 10.

4. The truncated tropomyosin according to claim 3, characterized in that When the tropomyosin is Pen a1, the amino acid sequence of the truncated tropomyosin is shown as SEQ ID NO. 1-3.

5. The truncated tropomyosin according to claim 3, characterized in that When the tropomyosin is Der p10, the amino acid sequence of the truncated tropomyosin is shown in SEQ ID NO. 4-6.

6. A nucleic acid molecule, characterized in that Used for expressing the truncated tropomyosin according to any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, characterized in that The nucleic acid molecule is codon-optimized.

8. A recombinant expression vector, characterized in that: Comprising the nucleic acid molecule according to claim 6 or 7 and a Bla g2 signal peptide gene.

9. A recombinant bacterium, characterized in that Comprising the nucleic acid molecule according to claim 6 or 7 or the recombinant expression vector according to claim 8.

10. Use of the truncated tropomyosin according to any one of claims 1 to 5 in the preparation of an allergen diagnostic kit or an anti-allergic vaccine.

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