CN112458093A

CN112458093A - Encoding gene of grapevine oyster allergenic protein TM and application thereof

Info

Publication number: CN112458093A
Application number: CN202011179694.1A
Authority: CN
Inventors: 刘光明; 云肖; 李梦思; 桓霏; 陈一瑜; 刘红
Original assignee: Jimei University
Current assignee: Jimei University
Priority date: 2020-10-29
Filing date: 2020-10-29
Publication date: 2021-03-09

Abstract

The invention provides a coding gene of a grapevine oyster sensitizing protein TM and application thereof, wherein the amino acid sequence of the TM of the sensitizing protein is shown as SEQ ID NO: 1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 2, respectively. According to the encoding gene of the ostrea grapevine allergenic protein, the gene is cloned into a prokaryotic expression vector, recombinant expression plasmids are constructed, host bacteria are transferred and induced to express, the recombinant ostrea grapevine allergenic protein TM can be obtained, the obtained amino acid sequence is applied to epitope prediction, and the recombinant ostrea grapevine allergenic protein TM and the serum of a patient with grapevine allergy are used for measuring the IgE binding capacity, so that a reference basis can be provided for clinical diagnosis of allergic symptoms and detailed screening of allergens, and the prevention, control and treatment of allergic diseases aiming at the TM are timely and accurate.

Description

Encoding gene of grapevine oyster allergenic protein TM and application thereof

Technical Field

The invention belongs to the technical field of food detection, and particularly relates to a gene for encoding grapevine oyster allergenic protein TM and application thereof.

Background

The statistical data of Chinese fishery in recent years show that the annual consumption of bivalve shellfish aquatic products such as oysters, variegated clams, blood clams, clams and the like is gradually increased year by year. Compared with deep crustacean aquatic products, related researches on allergens in shellfish aquatic products are few, and clinical diagnosis of shellfish food allergy is severely restricted.

Tropomyosin (TM) has a molecular weight of about 35-38 kDa, an isoelectric point of about 4.5, and thermal stability. The structure of the catalyst is composed of two methylene groups, each methylene group is in an alpha helical structure and is mutually wound to form a super-helical structure; ishikawa et al first chromatographically separated two allergens in Pacific oyster and designated Cra g 1 and Cra g 2. Leung, P.S. and the like carry out cDNA cloning and molecular identification on TM in Pacific oyster, a cloned product is named Cra g 1.03 and is successfully expressed to obtain 59kDa recombinant protein which can react with IgE antibodies of oyster allergy patients. Fang, l, et al identified immunodominant epitopes of pacific oyster TM (Cra g 1) and key amino acids for IgE binding. At present, the research on TM in Pacific oyster is complete, but as a novel allergen in the Ostrea vittata, the related information such as protein structure, epitope and immunological binding activity of TM is not deeply researched.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the art described above. Therefore, the first objective of the invention is to provide a gene for encoding the Ostrea dentata hypersensitive protein TM.

The second purpose of the invention is to propose a recombinant plasmid.

The third purpose of the invention is to provide a method for preparing the recombinant grapevine oyster allergenic protein TM.

The fourth purpose of the invention is to provide a recombinant grapevine oyster allergenic protein TM.

The fifth purpose of the invention is to provide the application of the ostrea grapevine allergenic protein.

In the first aspect of the present invention, there is provided a gene encoding a crassostrea viticola sensitizing protein TM, wherein the amino acid sequence of the crassostrea viticola sensitizing protein TM is as shown in SEQ ID NO: 1, and the nucleotide sequence of the gene is shown as SEQ ID NO: 2, respectively. According to the embodiment of the invention, the Ostrea dentata allergic protein TM is provided for the first time, the gene of the protein is connected with an expression vector, then the protein is transferred into an expression host and induced to express, and the Ostrea dentata allergic protein TM is obtained after purification; performing antigen epitope prediction analysis on the obtained TM amino acid sequence; in addition, the analysis of the binding capacity of the ostrea grapevine allergenic protein TM and the ostrea grapevine allergic patient serum IgE can provide convenience for component screening and clinical diagnosis of the ostrea grapevine allergic patient allergen.

In a second aspect of the present invention, there is provided a recombinant plasmid comprising the above-mentioned gene encoding a pacific protein TM of crassostrea vitis.

In a third aspect of the present invention, according to an embodiment of the present invention, there is provided a method for preparing recombinant pacifier oyster sensitizing protein TM, comprising the steps of:

(1) extracting total RNA from the muscular tissue of the portuguese oyster, and performing reverse transcription to obtain cDNA;

(2) carrying out PCR amplification by taking cDNA as a template to obtain the coding gene of the allergic protein of the ostrea grapevine;

(3) cloning the coding gene obtained in the step (2) into a prokaryotic expression vector to construct a recombinant plasmid;

(4) and transferring the recombinant plasmid into host bacteria, inducing the host bacteria to express and purify protein, and thus obtaining the recombinant concha avenae allergenic protein.

In a fourth aspect of the present invention, according to the embodiments of the present invention, the present invention also provides a recombinant ostrea grapevine sensitizing protein TM, which is prepared by the above-mentioned method for preparing recombinant ostrea grapevine sensitizing protein TM.

In a fifth aspect of the present invention, the present invention also proposes the use of the above-mentioned pacific protein TM of pacific oyster.

The first application is the application of prediction of antigen epitope among allergens of different species in amino acid sequences of the oyster sensitizing protein TM.

The second application is the application of the oyster hypersensitive protein TM in screening of allergen components TM.

According to the embodiment of the invention, the pacific oyster sensitizing protein TM is used for clinical immunoblot detection, ELISA detection or basophil activation test.

According to the embodiment of the invention, the application of the conching protein TM of the Crassostrea vittaria in screening of the allergen component TM can diagnose and screen the allergen component TM causing allergic symptoms of a patient to be the Viterocarpaea vittaria allergen, and is beneficial to prevention, control and treatment of allergic diseases of the TM by the patient.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is agarose gel electrophoresis of a grapevine oyster sensitizing protein TM gene;

FIG. 2 is SDS-PAGE analysis and Western-blot verification of sensitized protein TM of Ostrea dentata;

FIG. 3 is epitope analysis of Ostrea dentata sensitization protein TM;

FIG. 4 is the Dot-blot analysis of the protein TM of the oyster Portuguese allergic reaction and the serum of the oyster Portuguese allergic reaction patients.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The following disclosure provides many different embodiments, or examples, for implementing different embodiments of the invention. To simplify the disclosure, specific embodiments or examples are described below. Of course, they are merely examples and are not intended to limit the present invention. In addition, the present invention provides examples of various specific processes and materials, and one of ordinary skill in the art will recognize the applicability of other processes and/or the use of other materials. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, and the like, which are within the capabilities of persons skilled in the art. In addition, unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction and amino acid sequences are written from left to right in the amino terminus to carboxy terminus direction herein.

The invention is described below by way of illustrative specific examples, which do not limit the scope of the invention in any way. Specifically, the following are mentioned: the reagents used in the present invention are commercially available unless otherwise specified.

Example 1 cloning of recombinant Ostrea dentata sensitizing protein TM

(1) Design and Synthesis of primers

Related gene sequences of TM were searched in GenBank of National Center for Biotechnology Information, NCBI, the United states, Pacific oyster TM sequence (ID: KY549366.1) was selected, and specific primers were designed and synthesized using Primer Premier 5.0 software.

Primers for grape-tooth oyster TM gene cloning

TM-F：5’-ATGGACAGCATCAAGAAGAAGATGA-3’(SEQ ID NO：3)；

TM-R：5’-TTAATATCCTGCCAGCTCGGCAAAA-3’(SEQ ID NO：4)

(2) Cloning vector construction

Firstly, taking fresh oyster muscle of portugal oyster, and extracting total RNA of the oyster muscle according to a total RNA extraction kit; cDNA was synthesized using the method described in the Tiangen reverse transcription kit, and the reaction system is shown in Table 1.

TABLE 1 reverse transcription reaction System

PCR amplification is carried out by using the obtained cDNA as a template, the PCR reaction system is shown in Table 2, and the PCR amplification conditions are shown in Table 3, so as to obtain the encoding gene fragment (SEQ ID NO: 2) of the Ostrea meretrix TM.

TABLE 2 PCR reaction System

TABLE 3PCR amplification conditions

The PCR products were separated by electrophoresis on a 1% agarose gel, as shown in FIG. 1. The band M is DL2000 DNAmarker, and the bands 1-3 are the PCR amplification products of the TM full-length sequence. And (3) cutting and recovering the amplified product by adopting a DNA purification and recovery kit, and using the recovered product to construct a cloning vector. The recovered product (target gene) was ligated with the pEASY-T1 cloning vector using a 5. mu.L system, i.e., 4. mu.L of the recovered product and pEASY-T11. mu.L. After fully and uniformly mixing, the sample is placed at 25 ℃ for connection for 10min to obtain a connection product.

Transferring the obtained ligation product into a just thawed escherichia coli Trans T1 host, and carrying out ice bath for 30 min; the mixture was heat-shocked with 42 ℃ water bath for 30s and rapidly ice-cooled for 2 min. Adding 250 μ L LB culture medium into the heat-activated host cells, and culturing (37 ℃, 200rpm) for 1 h; the cells were plated on LB solid medium containing kanamycin sulfate (Kana) and cultured overnight at 37 ℃ in an inverted state.

And selecting a single colony on the overnight-cultured plate for colony PCR verification, and selecting a clone with correct PCR product molecular weight, single band and brightness for sequencing.

(3) Construction of expression vectors

Inoculating correctly sequenced clone to LB liquid culture medium containing 1mmol/L Kana, inoculating Escherichia coli expression vector pET-22b host to LB liquid culture medium containing 1mmol/L ampicillin (Amp), placing at 37 deg.C, shaking at 200rpm to OD₆₀₀And (3) centrifuging to 0.6-0.8 (12000g, 1min) to recover the thalli, and extracting plasmids according to the instructions of a small root plasmid extraction kit.

Carrying out double enzyme digestion on the positive clone and the pET-22b host plasmid by Nde I and Sal I, wherein the enzyme digestion system is carried out at 37 ℃ as shown in Table 4, the selected enzyme digestion time is 2h, after the enzyme digestion is finished, the enzyme digestion product is separated by electrophoresis of 1% agarose gel, and the enzyme digestion product is used for recovering a vector and a target gene fragment by using a Universal DNA purification recovery kit for constructing an expression vector.

The enzyme cutting site primer is as follows:

Nde I-F：5’-CATATGATGGACAGCATCAAGAAGA-3’(SEQ ID NO：5)；

Sal I-R：5’-GTCGACATATCCTGCCAGCTCGGCA-3’(SEQ ID NO：6)。

TABLE 4 double digestion reaction System

The enzyme digestion products are mixed according to the target fragment of 1 mu L, the enzyme digestion fragment of pET-22b of 4 mu L and the Solution I of 5 mu L, and are connected for 16h at the temperature of 16 ℃. The ligation product was transferred to E.coli expression host E.coli BL21(DE3) competent cells in the same manner and verified by colony PCR. And selecting a clone with correct PCR product molecular weight, single band and brightness for sequencing.

Example 2 preparation of Ostrea dentata recombinant TM protein

(1) Inducible expression of Ostrea vittata (TM)

The positive strains with correct sequencing are selected and inoculated into 20mL LB culture solution containing 1mmol/L Amp, and cultured overnight at 37 ℃. The next day, 5mL of the above culture solution was transferred to 500mL of LB culture solution containing 1mmol/L Amp at a ratio of 1:100,culturing at 37 deg.C to OD₆₀₀Adding isopropyl thiogalactoside (IPTG) with the final concentration of 1mmol/L, inducing at 16 ℃ for 16h, centrifuging (12000g, 20min, 4 ℃), and collecting thalli; resuspending in 30mL of ice-chilled lysis buffer (20mmol/L Tris-HCl, 200mmol/L NaCl, pH 8.0), sonicating the cells, recentrifugation (12000g, 30min, 4 ℃), collecting the supernatant and subjecting it to Ni²⁺Purification on an NTA affinity column (1.0 cm. times.5.0 cm).

(2) Purification and identification of oyster TM from Portugal teeth

The supernatant after disruption and centrifugation was applied to a Ni column equilibrated in advance with an equilibration solution (20mmol/L Tris-HCl, 0.2mol/L NaCl, 10mmol/L imidazole, pH 8.0) at a rate of 1 mL/min. After the sample loading is finished, the balance solution is used to wash off the non-combined hybrid protein, and the eluent (20mmol/L Tris-HCl, 0.2mol/L NaCl, 100mmol/L imidazole, pH 8.0) is used to elute the target protein until the determination A is finished₂₈₀The value was 0.05 or less, and then SDS-PAGE analysis was performed on the eluted fractions and Western-blot analysis was performed using rabbit anti-Ostreidae TM antibody (dilution 1: 5000) as the primary antibody.

The preparation method of the rabbit anti-Ostrea vittata TM antibody comprises the following specific steps: the purified Crassostrea vitis TM was concentrated to 1mg/mL immunized New Zealand white rabbits (body weight approx. 2 kg). In the primary immunization, TM is fully mixed with equivalent volume of Freund's complete adjuvant and then is injected subcutaneously; after two weeks intervals TM was mixed well with an equal volume of incomplete freund's adjuvant and rabbits were boosted. After three times of boosting immunity, taking a small amount of rabbit ear arterial serum to detect the titer; if the titer does not meet the requirement, the impact immunization is carried out for 1 time, and carotid blood is collected after 5 days. Standing the obtained blood at 4 deg.C for at least 4h, centrifuging at 4 deg.C 8000 Xg for 15min, collecting serum, packaging, and freezing at-20 deg.C. The immunization process is finished by the experimental animal center of the college of pharmacy of Xiamen university.

Collecting the purified part to obtain the oyster TM. As shown in FIG. 2, the Ostrea vittata (TM) was expressed in large amounts, and after washing off the contaminating proteins from the equilibrium stream, it was eluted in an eluent containing 100mM imidazole, yielding a single band after purification (FIG. 2A). Western-blot results show that the purified protein component and the rabbit anti-Ostrea vittata TM polyclonal antibody have specific bands, and further show that the purified protein is Ostrea vittata TM. In the figure, the band M is a standard protein, the band 1 is a protein sample before loading, the bands 2-3 are flow-washing samples, and the bands 4-9 are purified proteins collected by elution.

Example 3 epitope prediction analysis of Crassostrea viticola recombinant protein TM

Based on the physicochemical properties of the TM amino acid sequence, four kinds of immuno-informatics software, DNAstar protein, Bepided 1.0server, ABCPred and Immunodicine Group, were used to perform predictive analysis on the epitope of Ostrea vinifera (TM), and the results are shown in FIG. 3. Selecting a sequence jointly predicted by three or more kinds of software as a predicted epitope, and finally obtaining 3 more key linear epitope regions on the TM amino acid sequence: 15-47, 61-85, 210-245.

Example 4 Portugal oyster recombinant protein TM and Portugal oyster allergic patient serum Dot blot analysis

Separately, 2. mu.L of the recombinant TM and BSA obtained in example 2 at a concentration of 1mg/mL were spotted on a nitrocellulose membrane (0.7X 0.7cm per cell), and left to dry; soaking the dried nitrocellulose membrane in 5% skimmed milk, and incubating for 1.5h at room temperature on a shaking table; the nitrocellulose membrane after blocking was washed 3 times for 5min each with TBST (17g NaCl, 40mL 1M Tris-HCl, pH 8.0, 1mL Tween-20 in 2L distilled water). Respectively placing the nitrocellulose membranes in serum of a patient allergic to the grapevine oyster diluted by TBST at a ratio of 1:1, and placing the serum on a shaking table for incubation at room temperature overnight; taking out the nitrocellulose membrane soaked in the serum diluent every other day, washing with TBST for 5 times, 5min each time, and incubating with goat anti-human IgE antibody labeled with horseradish peroxidase diluted by TBST at a ratio of 1:10000 for 1.5h at room temperature on a shaking table; after incubation is finished, washing the membrane for 7 times by using TBST, wherein each time is 7min, placing the membrane in a developing solution, incubating the membrane for 2min at room temperature in a dark place, taking out the membrane, and taking a picture by using a chemiluminescence fluorescent gel imager; as shown in FIG. 4, the results of the test were shown in the figures, in which Nos. 1, 2, 3, 4 and 5 were serum from patients with allergy to Ostrea dentata, and NS was serum from patients with non-allergy, and was used as a negative control group. As can be seen from the figure, when purified P.vinifera recombinant protein TM binds to specific IgE in the serum of patients with P.vinifera allergy, the component of the allergen causing the allergic symptoms in these patients is TM.

In summary, according to the embodiments of the present invention, the grapevine oyster sensitizing protein TM can be obtained; according to the invention, the encoding gene of the grapevine oyster allergenic protein is cloned into a prokaryotic expression vector, recombinant expression plasmids are constructed, and then host bacteria are transferred and induced to express the recombinant grapevine oyster allergenic protein TM, and the epitope of the recombinant grapevine oyster allergenic protein TM can be predicted by combining various immune informatics software according to the amino acid sequence of the grapevine oyster allergenic protein TM; the allergenic protein can be combined with specific IgE of the serum of a patient allergic to the ostrea vittata, so that the allergenic protein can be used for detecting allergenic components and is beneficial to preventing and controlling allergic diseases caused by ostrea vittata TM.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Sequence listing

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Val Gln Asn Asp Gln Ala Ser Gln Arg Glu Asp Ser Tyr Glu Glu Thr

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gaagatctca catcattgca gaaaaagcac tccaacttag agaatgagtt cgatacagtg 180

aacgagaaat accaggaatg ccaaaccaaa atggaggagg cagaaaagac cgcttcagag 240

gcggaacaag agatccagag tttgaaccgc aggatccagc tcctagagga agacatggag 300

aggtcggagg agcggctcca gaccgccacc gagaagttgg aggaggcttc caaagccgca 360

gacgagagcg agagaaaccg caaggtcttg gaaaacctca ataacgcctc agaggagaga 420

actgatgtct tagagaaaca gcttactgag gccaagctga tcgctgagga ggccgacaaa 480

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Claims

1. The encoding gene of the Ostrea dentata sensitization protein TM is characterized in that the amino acid sequence of the Ostrea dentata sensitization protein TM is shown as SEQ ID NO: 1, and the nucleotide sequence of the gene is shown as SEQ ID NO: 2, respectively.

2. A recombinant plasmid comprising the coding gene according to claim 1.

3. A method for preparing recombinant Ostrea vittata allergic protein TM is characterized by comprising the following steps:

(2) performing PCR amplification by using cDNA as a template to obtain a coding gene of the sensitized protein of the oysters grapevine according to claim 1;

(4) and transferring the recombinant plasmid into host bacteria, inducing the host bacteria to express and purify protein to obtain the recombinant grapevine oyster allergenic protein TM.

4. A recombinant Ostrea dentata sensitization protein TM, characterized in that it is prepared by the method for preparing recombinant Ostrea dentata sensitization protein TM according to claim 3.

5. Use of the pacific oyster sensitiser TM according to claim 4 for the prediction of epitopes between allergens of different species.

6. Use of the pacifier oyster allergenic protein TM according to claim 4 in screening of allergenic component TM.

7. The use according to claim 6, wherein the Crassostrea grapevines sensitizing protein TM is used in a clinical immunoblot assay, ELISA assay or basophil activation assay.