Coding gene of scylla paramamosain allergen troponin C and application thereof

Abstract

The invention provides a coding gene of scylla paramamosain allergen troponin C and application of the coding gene. The amino acid sequence of the allergen is shown as SEQ ID NO: 1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 2. The method comprises the following steps: constructing a recombinant expression plasmid of a scylla paramamosain troponin C (TnC) coding gene, transferring the recombinant expression plasmid into a host cell, inducing expression, and purifying to obtain the recombinant scylla paramamosain sensitization protein TnC. The TnC can be combined with IgE in the serum of a crustacean allergy patient, the expression of CD63 and CD203c on the basophilic granulocyte surface in the whole blood of the patient can be remarkably up-regulated, and the sensitizer can be prepared into a diagnostic reagent for sensitizing components in a patient serum sample or a desensitizing preparation, and has a wide application prospect in the fields of diagnosis and treatment of allergic diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and in particular relates to a coding gene of mud crab allergenic troponin C and an application thereof. Background Art

[0002] Scylla paramamosain is a crustacean aquatic product with the advantages of fast growth, delicious meat and high nutritional value. It is one of the important economic aquatic products in the southeast coast of my country. However, the problem of food allergies induced by it cannot be ignored.

[0003] At present, the allergens in Scyphus pseudoburrowing crab that can be recognized by the serum specific immunoglobulin E of patients with crab allergy include: tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium binding protein, triosephosphate isomerase, and filament protein C. These allergens are named Scy p 1, Scy p 2, Scy p 3, Scy p 4, Scy p 8, and Scy p 9. Among them, there is still a lack of reports on troponin C (Troponin C, TnC) in crabs, so improving the immunological characteristics of Scyphus pseudoburrowing crab TnC will help to fully understand the allergenicity of Scyphus pseudoburrowing crab. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems in the above-mentioned technology to a certain extent. To this end, the present invention aims to provide a gene encoding troponin C sensitizing agent of mud crab and its application.

[0005] In the first aspect of the present invention, a Scylla sylvaticum allergen TnC is provided, the amino acid sequence of which is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding the Scylla sylvaticum allergen TnC is shown in SEQ ID NO: 2.

[0006] The mud crab allergen TnC of the present invention has a full length of 453bp in its coding gene, and the mud crab allergen TnC is proposed for the first time. The mud crab allergen TnC is used to carry out IgE binding test and basophil activation test with the serum and whole blood of patients with crab allergy, and the binding ability and immunoreactivity of the mud crab allergen TnC are analyzed, so that the mud crab allergen TnC can be used to prepare diagnostic reagents or desensitization preparations for crab allergic diseases.

[0007] In the second aspect of the present invention, a recombinant plasmid is provided, comprising a gene encoding the above-mentioned Scylla sphaeroides allergen TnC.

[0008] In the third aspect of the present invention, a method for preparing the mud crab allergen TnC is provided, which comprises the following steps:

[0009] (1) using the cDNA of Scylla pseudoburrowing as a template and the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers to perform PCR amplification to obtain a gene encoding the Scylla pseudoburrowing allergen TnC;

[0010] (2) The coding gene obtained in step (1) is cloned into a prokaryotic expression vector to construct the above-mentioned recombinant plasmid, which is then transferred into a host cell, and a single colony with correct sequencing is selected for activation and induction of expression and purification to obtain the TnC protein product of the mud crab.

[0011] In the fourth aspect of the present invention, the present invention also proposes the use of the above-mentioned mud crab allergen TnC in the preparation of diagnostic reagents or desensitization preparations for crab allergic diseases.

[0012] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The recombinant allergenic protein TnC of mud crab is cloned, expressed and purified, wherein A is the agarose gel electrophoresis analysis of the full-length gene of the allergenic protein TnC of mud crab, and B is the SDS PAGE analysis of the purified protein of the allergenic protein TnC of mud crab;

[0014] Figure 2 This is the mass spectrometry identification of the recombinant mud crab allergenic protein TnC, wherein A is the secondary mass spectrum of the mud crab allergenic protein TnC, and B is the mass spectrum peptide matching diagram of the mud crab allergenic protein TnC;

[0015] Figure 3 To analyze the IgE binding activity of the recombinant mud crab allergenic protein TnC with the sera of patients with crab allergy, including 10 patients with crab allergy and 3 healthy subjects;

[0016] Figure 4 This is a basophil activation test with the mud crab allergenic protein TnC. A is the flow cytometer plot of the basophil activation experiment (white peak: PBS-stimulated cell population, blue and red peaks: mud crab allergenic protein TnC-stimulated cell population, a total of 7 crab allergy patients and 3 healthy subjects), B is the analysis of the ratio of CD63-positive cells on the surface of basophils, and C is the analysis of the ratio of CD203c-positive cells on the surface of basophils. DETAILED DESCRIPTION

[0017] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0018] The disclosure below provides many different embodiments or examples to realize different implementation methods of the present invention. In order to simplify the disclosure of the present invention, specific embodiments or examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the examples of various specific processes and materials provided by the present invention, those of ordinary skill in the art can be aware of the applicability of other processes and / or the use of other materials. Unless otherwise stated, the implementation of the present invention will adopt the traditional techniques in the fields of chemistry, molecular biology, etc. within the capabilities of those skilled in the art. In addition, unless otherwise stated, in this article, nucleic acids are written from left to right in the direction of 5' to 3', and amino acid sequences are written from left to right in the direction of amino terminal to carboxyl terminal.

[0019] The present invention is described below by way of illustrative specific examples, which are not intended to limit the scope of the present invention in any way. It is particularly noted that the reagents used in the present invention are commercially available unless otherwise specified.

[0020] Example 1 Cloning of the allergenic protein TnC of Scylla pseudoburrows

[0021] 1. Extraction of Total RNA

[0022] according to The total RNA of Scylla pseudoburrowing was extracted according to the instructions of Super Total RNA Extraction Kit and stored at -80℃ for later use after testing its concentration and purity.

[0023] 2. Synthesis of cDNA

[0024] According to the instructions of PrimeScriptTMII 1st Strand cDNA Synthesis Kit, total RNA of Scylla pseudoburrows was reverse transcribed into cDNA and stored at -80°C for future use after its concentration and purity were detected.

[0025] 3. Primer Design and Synthesis

[0026] The relevant gene sequences of Scylla pseudoburrowingii TnC were retrieved from GenBank of the National Center for Biotechnology Information (NCBI) database, and specific primers were designed and synthesized using Primer Premier 5.0 software (Table 1).

[0027] Table 1 Primers used for cloning the TnC gene of Scylla parasitica

[0028]

[0029] 4. Full-length gene amplification

[0030] The cDNA of the above-mentioned mud crab TnC was used as a template, and TnC-F (SEQ ID NO: 3) and TnC-R (SEQ ID NO: 4) in Table 1 were used as primers for PCR amplification. The amplified product was analyzed by agarose gel electrophoresis. The results are as follows: Figure 1 As shown in A, the coding gene fragment of the allergenic protein TnC of the mud crab was obtained (SEQ ID NO: 2). The PCR reaction system is shown in Table 2, and the PCR amplification conditions are shown in Table 3;

[0031] Table 2 PCR reaction system

[0032]

[0033] Table 3 PCR amplification conditions

[0034]

[0035] 5. Vector Construction

[0036] The full-length gene was recovered using a DNA purification recovery kit. The full-length gene was connected to the pEASY-T1 vector at 25°C for 15 minutes and transformed into Trans-T1 competent cells. After colony PCR verification, a positive single colony was picked for sequencing. The clones and pET-32a expression vectors with correct sequencing were activated and the plasmids were extracted. The plasmids were further double-digested with restriction endonucleases Nde I and Sal I. The digested pET-32a expression vector and target gene fragment were recovered using a DNA purification recovery kit. The target fragment was connected to the pET-32a digested fragment at 16°C for 16 hours, and the ligation product was transferred to E.coli Rosetta-gami 2 (DE3) competent cells. After colony PCR verification, a positive single colony was picked for sequencing.

[0037] Example 2 Expression, purification and identification of the allergenic protein TnC of Scylla pseudoburrows

[0038] 1. Induced expression and purification of TnC in Scylla pseudoburrows

[0039] The positive bacteria with correct sequencing were inoculated into LB liquid medium and cultured at 37°C until OD 600=0.6-0.8, add the inducer IPTG with a final concentration of 0.5mmol / L, induce at 37℃ for 4h, then centrifuge to collect the cells; resuspend in pre-cooled ultrasonic buffer (20mmol / L Tris-HCl, 0.2mol / L NaCl, pH 8.0) for ultrasonic disruption, collect the supernatant after centrifugation, load on Ni balanced in Buffer A (20mmol / L Tris-HCl, 0.2mol / L NaCl, 10mmol / L imidazole, pH 8.0) 2+ -NTA affinity chromatography column, using Buffer A for washing and Buffer B (20mmol / L Tris-HCl, 0.2mol / LNaCl, 100mmol / L imidazole, pH 8.0) for elution, and SDS-PAGE analysis results are as follows Figure 1 As shown in B.

[0040] 2. Mass spectrometry identification of TnC in Scylla pseudoburrows

[0041] Cut Figure 1 The gel strip of the single target band in B, with a molecular weight of about 20 kDa, was sent to Shenzhen Micron Biotechnology Co., Ltd. for mass spectrometry identification; the identification results Figure 2 As shown, 59% of the isolated peptides were identified to match the amino acid sequence of the allergenic protein TnC of Scylla pseudoburrows.

[0042] Example 3 Application of the allergenic protein TnC of Scylla pseudoburrows in clinical diagnosis

[0043] 2 μg of mud crab allergenic protein TnC and BSA were spotted onto the nitrocellulose membrane, and after drying, it was blocked with 5% skim milk for 1.5 hours. After blocking, it was washed with TBST, and the nitrocellulose membrane was incubated overnight in the sera of crab allergy patients and healthy people diluted 1:1. The next day, it was washed with TBST and incubated with goat anti-human IgE antibody. After washing with TBST, it was developed with a chemiluminescence imager. Figure 3 As shown, it was found that the mud crab allergenic protein TnC can bind to the IgE of patients with crab allergies, indicating that the mud crab allergenic protein TnC obtained in the present application has IgE binding ability.

[0044] The basophil activation test was performed by stimulating the whole blood of crab allergy patients and healthy subjects with the allergenic protein TnC of the mud crab. The specific steps are as follows: take out the whole blood of crab allergy patients or healthy subjects from the heparin anticoagulation tube, add PBS and the allergenic protein TnC of the mud crab for stimulation, and use fluorescent labeled antibodies (anti-human CD63, anti-humanCD203c, anti-human HLA-DR) to stain in the dark, and analyze on a flow cytometer. Compared with cells stimulated with PBS, the activation of basophils was mainly based on CD63. + Cells and CD203c + The percentage of cells is shown. Figure 4 As shown, the mud crab allergenic protein TnC can stimulate the expression of CD63 and CD203c on the surface of basophils in the whole blood of patients with crab allergy, indicating that the mud crab allergenic protein TnC obtained in the present application has immunoreactivity.

[0045] In summary, according to the present invention, the coding gene of the mud crab allergenic protein TnC is cloned into a prokaryotic expression vector, a recombinant expression plasmid is constructed, and then transferred into a host bacterium and induced to express, so as to obtain the mud crab allergenic protein TnC. The mud crab allergenic protein TnC is used to measure the immunological activity of TnC with the serum and whole blood of patients with crab allergy, which can provide a reference for the clinical diagnosis of allergic symptoms and the detailed screening of allergens, and can be used for the preparation of diagnostic reagents for crab allergic diseases, and can also be used for the preparation of crab desensitization preparations to improve the desensitization efficacy of crab.

[0046] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0047] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A troponin C sensitized by mud crab, characterized in that: The amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding the mud crab troponin C is shown in SEQ ID NO:

2.

2. A recombinant plasmid, characterized in that: It comprises a gene encoding the mud crab allergenic troponin C according to claim 1.

3. A method for preparing troponin C sensitized by Scylla pseudoburrows, characterized in that: The following steps are involved: (1) using the cDNA of Scylla pseudoburrows as a template and the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers to perform PCR amplification to obtain the gene encoding the Scylla pseudoburrows allergenic troponin C described in claim 1; (2) The coding gene obtained in step (1) is cloned into a prokaryotic expression vector to construct the recombinant plasmid described in claim 2, and the recombinant plasmid is transferred into a host cell, and a single colony with correct sequencing is selected for activation and induction of expression and purification to obtain the troponin C protein product of the pseudo-crab.

4. Use of the mud crab allergenic troponin C as claimed in claim 1 in the preparation of diagnostic reagents or desensitization preparations for crab allergic diseases.

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