CN115963273A - Application of portunus trituberculatus nSCP or rSCP and kit - Google Patents

Abstract

The invention provides application and a kit of Portunus trituberculatus nSCP or rSCP, belonging to the technical field of allergen detection, wherein the nucleotide sequence of nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of rSCP is shown as SEQ ID NO. 2. The invention enriches the types of deficient portunus trituberculatus allergen, can improve the diagnosis and immunotherapy level related to crab allergic diseases, and can improve the detection accuracy and specificity of the crab allergen in food by utilizing the detection limit of the portunus trituberculatus troponin when detecting the calcium-binding protein of the portunus trituberculatus.

Description

Application of portunus trituberculatus nSCP or rSCP and kit

Technical Field

The invention belongs to the technical field of allergen detection, and particularly relates to application of portunus trituberculatus nSCP or rSCP and a kit.

Background

Crustaceans are one of eight major types of allergic foods proposed by food and agriculture organization and world health organization in the united nations, wherein crab allergy is one of the most common causes of food allergy in asian areas such as china, japan and thailand. In view of the economic size and the allergic incidence of the blue crabs, the method has important economic significance and clinical significance for researching the allergens of the blue crabs.

But the research on the blue crab allergen is quite insufficient at present. There are no Portunus trituberculatus allergen protein at the World Health Organization (WHO) and the International Union of Immunological Societies (IUIS) licensed allergen nomenclature Committee website (http:// www. Allergen. Org /). Due to the fact that the types of the portunus trituberculatus allergen sequences are not comprehensive, and the properties such as allergenicity and the like are not clear, a reasonable detection standard product is not available for accurately detecting food allergens at present, and a comprehensive and proper diagnostic preparation is also not available for accurately diagnosing allergic patients. Therefore, the systematic identification and research of the portunus trituberculatus allergens are carried out, each allergen protein is known, the properties of each allergen protein are utilized to carry out the application of compounding or single-component of various allergens, and the stable and accurate detection and diagnosis results are obtained urgently.

The Sarcoplasmic calcium binding protein (SCP) has the characteristics of high heat and acid-base stability and strong species specificity, and has the potential for allergen detection and diagnosis. But no research on the blue crab SCP allergy is found at present. Due to sequence differences in SCP between species, its epitopes are different. Therefore, other species of SCP sequences cannot be used in place of portunus trituberculatus SCP for food detection or allergy diagnosis, which may cause false detection and missed detection, false diagnosis or failure to identify the allergy of the patient to portunus trituberculatus, and cause additional economic burden and life pressure of the patient.

Disclosure of Invention

In view of the above, the invention aims to provide an application and a kit of portunus trituberculatus nSCP or rSCP, which are used for solving the problems that the portunus trituberculatus allergen in food cannot be accurately and specifically detected, and the allergic diseases caused by portunus trituberculatus cannot be accurately diagnosed.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides an application of blue crab nSCP or rSCP in preparing a reagent for diagnosing allergic diseases, wherein the nucleotide sequence of nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of rSCP is shown as SEQ ID NO. 2.

Preferably, the allergic disease is caused by portunus trituberculatus.

The invention provides an application of blue crab nSCP or rSCP in preparing a food allergen detection reagent, wherein the nucleotide sequence of nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of rSCP is shown as SEQ ID NO. 2.

The invention provides an ELISA kit for detecting Portunus trituberculatus SCP allergen, which comprises Portunus trituberculatus SCP; the SCP comprises nSCP or rSCP, wherein the nucleotide sequence of nSCP is shown in SEQ ID NO. 1, and the nucleotide sequence of rSCP is shown in SEQ ID NO. 2.

Preferably, the kit also comprises a coating diluent, a sealing solution, a washing solution, a developing solution, a stopping solution and a negative quality control substance.

Preferably, the negative quality control product is human serum.

Preferably, the preparation method of the rSCP comprises the following steps:

the His tag sequence, the enterokinase site sequence and the blue crab nSCP sequence are inserted into a pET-30a plasmid to construct a recombinant plasmid, and then the recombinant plasmid is transformed into a competent cell to carry out induction expression.

Preferably, the sequence of the His tag is shown as SEQ ID NO. 3, the sequence of the enterokinase site is shown as SEQ ID NO. 4, and the sequence of the Portunus trituberculatus nSCP is shown as SEQ ID NO. 1.

The preparation method of rSCP also comprises column chromatography purification, and the column chromatography purification method comprises the following steps:

centrifuging and crushing the fermentation liquor for induction expression to obtain a supernatant, adding the supernatant into a gravity column for nickel affinity purification, and washing with a binding buffer solution, a cleaning solution and an eluent in sequence to obtain rSCP.

Preferably, the binding buffer comprises 45-55mM Tris, 280-320 mM NaCl;

the cleaning solution comprises 45-55mM Tris, 280-320mM NaCl and 15-25 mM imidazole;

the eluent comprises 45-55mM Tris, 280-320mM NaCl and 490-510 mM imidazole.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a troponin in portunus trituberculatus as an allergen for the first time, portunus trituberculatus nSCP or rSCP is used for detecting allergic patients, and the portunus trituberculatus nSCP or rSCP has stronger IgE binding capacity with serum of the allergic patients. The method has the advantages that the detection limit of the portunus trituberculatus troponin during the detection is 0.29ng/mL, the quantification limit is 0.77ng/mL, and the cross reaction rate is low, so the method enriches the types of the deficient portunus trituberculatus allergens, can improve the diagnosis and immunotherapy level related to the crab allergic diseases, and can improve the detection accuracy and specificity of the crab allergens in food.

Drawings

FIG. 1 is an agarose gel electrophoresis of PCR amplification products of CDS gene of blue crab; in the figure: 1. DL 2000Plus DNA marker,2, PCR amplification product;

FIG. 2 is a diagram showing the effect of recombinant expression of Portunus trituberculatus rSCP; in the figure: 1. supernatant after cell disruption, 2, precipitation after cell disruption, 3, penetrating liquid, 4, washing liquid, 5, impurity washing liquid, 6 and eluent;

FIG. 3 is a graph showing the result of IgE reaction between Portunus trituberculatus nSCP and allergic patient serum; in the figure: A. indirect ELISA result graph, B, dot Blot result graph;

FIG. 4 is a graph showing the results of IgE reaction between Portunus trituberculatus rSCP and the serum of allergic patients;

FIG. 5 is a blue crab SCP sandwich ELISA standard curve;

FIG. 6 shows the results of the specificity evaluation of the sandwich ELISA.

Detailed Description

The invention provides application of blue crab nSCP or rSCP in preparing a reagent for diagnosing allergic diseases, wherein the nucleotide sequence of the nSCP is shown in SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown in SEQ ID NO. 2.

In the invention, the portunus trituberculatus sarcoplasmic calcium binding protein, namely portunus trituberculatus nSCP has higher sequence homology with other crab SCPs. Indirect ELISA or Dot Blot is carried out on the portunus trituberculatus nSCP or rSCP and the serum of an allergic patient to verify that the portunus trituberculatus SCP is really an allergen and the feasibility of the application of the portunus trituberculatus nSCP or rSCP in the aspect of detection and diagnosis. The allergic disease is preferably caused by blue crab.

The invention provides an application of blue crab nSCP or rSCP in preparing a food allergen detection reagent, wherein the nucleotide sequence of nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of rSCP is shown as SEQ ID NO. 2.

In the present invention, the food product may include products prepared from portunus trituberculatus, such as crab cream sauce, crab meat, crab stick, crab cream flour, crab cream crisps, crab cream crispy rice, and the like.

The invention provides an ELISA kit for detecting Portunus trituberculatus SCP allergen, which comprises Portunus trituberculatus SCP; the SCP comprises nSCP or rSCP, wherein the nucleotide sequence of nSCP is shown in SEQ ID NO. 1, and the nucleotide sequence of rSCP is shown in SEQ ID NO. 2.

In the invention, the portunus trituberculatus sarcoplasmic calcium binding protein is named as portunus trituberculatus nSCP, and the recombinant portunus trituberculatus sarcoplasmic calcium binding protein is named as portunus trituberculatus rSCP. The nucleotide sequence of the nccp is as follows:

atggcttactcttgggacaaccgcgtcaagtatgtcgtcaggtacatgtacgacatcgacaacaatggctacctcgacaagaatgactttgagtgcctggcgctgaggaacacactcatcgagggccgcggcgaattcaactccgacgcctatgccaacaaccagaagattatgagcaacctctggaacgagatcgccgagctggctgatttcaacaaggacggtcaggttactgtggacgagttcaaacaagccgtccagaagctgtgttgcggcaagaactttgatgctttccctccttgcttcaagactgtcattggtcacctgttcaagactattgatatcaacggtgacggccttgttggcgttgatgagtacaggctggattgcatctccaggagtgccttctcctccgttaaggagattgatgatgcttatggcaagctctgcactgacgatgacaagaaggctggtggcatcagcctcagccgttaccaggagctgtatgcccagttcatctccaaccctgacgagaagtgcaatgccgtctaccttttcggacccctgaaggaggtgcagtaa。

in the present invention, the nucleotide sequence of the rSCP is as follows:

atgcaccatcatcatcatcatgacgacgacgacaagatggcatatagctgggataaccgtgttaaatatgttgttcgttatatgtatgatatcgataacaacggttatctggataaaaacgattttgaatgtctggccctgcgtaatacgctgattgaaggtcgcggtgaatttaatagtgatgcatacgctaataaccagaaaattatgagtaacctgtggaatgaaatcgcagaactggccgattttaacaaagatggtcaggttaccgttgatgaatttaaacaggccgtgcagaaactgtgttgtggcaaaaattttgatgcgtttccgccttgttttaaaaccgttattggtcatctgtttaaaaccattgatatcaatggcgatggtctggttggcgttgatgaatatcgtctggattgtattagtcgtagtgcattttctagtgttaaagaaattgatgatgcctatggtaaactgtgtaccgatgatgataaaaaggcgggtggtattagtctgagtcgttatcaggaactgtatgcccagtttattagtaacccggatgaaaaatgtaacgcagtttatctgtttggtcctctgaaagaagtacagtaa。

in the invention, the kit also comprises coating diluent, confining liquid, washing liquid, developing liquid, stopping liquid and negative quality control substances. The coating diluent is preferably a CBS solution. The blocking solution is preferably PBST containing 0.5 to 6% of BSA. The wash solution is preferably PBST. The color developing solution is preferably a TMB single-component color developing solution. The stop solution is preferably 1.5 to 2.5M sulfuric acid. The negative quality control product is preferably human serum. The source of the CBS solution is not particularly limited in the invention, and the CBS solution can be prepared by using products sold in the field or conventional preparation methods.

In the present invention, the preparation method of the above-mentioned rSCP preferably comprises the steps of:

the His tag sequence, the enterokinase site sequence and the blue crab nSCP sequence are inserted into a pET-30a plasmid to construct a recombinant plasmid, and then the recombinant plasmid is transformed into a competent cell to carry out induction expression.

In the invention, the His label of the invention hardly influences the structure and the biological activity of the Portunus trituberculatus rSCP, and the protein of the Portunus trituberculatus rSCP is further utilized for purification. The His tag sequence is preferably shown as SEQ ID NO. 3, wherein the His tag sequence is caccatcatcatcatcatcat. In the invention, the enterokinase has the characteristics of high specificity and high hydrolysis efficiency, can be used as a substrate for specifically identifying and cutting enterokinase cutting sites by protease, and the preferred enterokinase site sequence is shown as SEQ ID NO. 4, wherein the enterokinase site sequence is gacgacgacgacaag; the preferred sequence of the Portunus trituberculatus nSCP is shown as SEQ ID NO. 1. In the present invention, if an rSCP completely identical to the nSCP sequence is to be obtained, a commercially available enterokinase product is selected, and the His tag sequence and the enterokinase site are excised according to the instructions of the product.

As a preferred embodiment, the preparation method of the rSCP further comprises column chromatography purification, and the method of column chromatography purification comprises:

centrifuging and crushing fermentation liquor for induced expression, taking supernatant, adding the supernatant into a gravity column for nickel affinity purification, and washing by using a binding buffer solution, a cleaning solution and an eluent in sequence to obtain rSCP.

In the present invention, the binding buffer is a buffer system in which rSCP is bound to the filler, and the binding buffer preferably includes 45-55mM Tris, 280-320 mM NaCl. The washing solution of the present invention, which preferably includes 45 to 55mM Tris,280 to 320mM NaCl,15 to 25mM imidazole, removes weakly bound hetero-proteins using a low concentration of imidazole. The eluent of the present invention competitively elutes the target protein using high concentration imidazole, and preferably includes 45-55mM Tris, 280-320mM NaCl, 490-510 mM imidazole. The rSCP purity in the eluent is calculated to be more than 97% by using Image J, so that the rSCP protein purity of the portunus trituberculatus obtained by the method is high.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Determination of CDS sequence of portunus trituberculatus sarcoplasmic calcium binding protein

(1) Total RNA extraction

Using a total RNA extraction kit (solibao, beijing), the total RNA extraction of the portunus trituberculatus muscle was performed according to the instruction procedures.

(2) Reverse transcription

Immediately after RNA extraction 5. Mu.L of RNA was digested with genomic DNA and reverse transcribed to synthesize cDNA using HiScript III RT Supermix for qPCR (+ gDNA wrapper) (Novozan, nanjing) according to the instructions, and the product was stored in aliquots at-20 ℃.

(3) Primer design and Synthesis

Selection of sequences outside the CDS region of the transcriptome primers were designed as follows, and the primer information is shown in Table 1.

TABLE 1 primer information

(4) PCR amplification

PCR amplification was performed using cDNA obtained by the above reverse transcription as a template and the above F/R as a primer, using 2X Rapid Taq Master Mix (Novovozam, nanjing).

PCR amplification reaction System: cDNA template 1.5. Mu.L, primers 2. Mu.L each, 2 × Rapid Taq Master Mix 25. Mu.L, ddH ₂ OμL。

PCR amplification reaction conditions: 35 cycles of 95 ℃ for 5min, then 95 ℃ for 15sec, 56 ℃ for 15sec, and 72 ℃ for 5sec, and finally 72 ℃ for 5min.

(5) Product validation

The PCR product amplified in step (4) was subjected to 1% agarose gel electrophoresis, and the results are shown in FIG. 1.

As shown in FIG. 1, the band of interest at about 600bp is bright and visible at a high occupancy rate, i.e., the band of interest is amplified.

(6) Sequencing analysis

The PCR amplification product is subjected to bidirectional sequencing by using primers F and R, positive and negative sequencing results are compared and spliced, an ATG-TAA sequence is selected to obtain a Portunus trituberculatus CDS sequence, as shown in SEQ ID NO:1, the sequence is compared with a reported crab SCP sequence, and the homology is more than 80 percent, so that the sequence can be determined as the SCP sequence.

Wherein the amino acid sequence of the Portunus trituberculatus SCP is shown as SEQ ID NO. 7, and the amino acid sequence is as follows:

MAYSWDNRVKYVVRYMYDIDNNGYLDKNDFECLALRNTLIEGRGEFNSDAYANNQKIMSNLWNEIAELADFNKDGQVTVDEFKQAVQKLCCGKNFDAFPPCFKTVIGHLFKTIDINGDGLVGVDEYRLDCISRSAFSSVKEIDDAYGKLCTDDDKKAGGISLSRYQELYAQFISNPDEKCNAVYLFGPLKEVQ。

example 2

A preparation method of Portunus trituberculatus rSCP comprises the following steps:

(1) Construction of recombinant plasmids

The His tag sequence (caccatcatcatcatcatcat), the enterokinase site (gacgacgacgacaag) and the Portunus trituberculatus SCP CDS sequence (SEQ ID NO: 1) are inserted between the plasmid enzyme cutting sites NdeI and XhoI of pET-30a through double enzyme digestion to construct a recombinant plasmid (Scophytum, beijing).

(2) Transformation and expression

Coli BL21 (DE 3) (Bio-Industrial, shanghai) cells were transformed with the recombinant plasmid according to the product instructions by chemical transformation, and single colonies were picked and cultured in LB liquid medium containing 50. Mu.g/mL kanamycin at 37 ℃ for 12h. And (5) preserving the strain of the bacterial liquid, performing bidirectional sequencing, and performing subsequent expression if the strain is consistent with the target gene. The strain was streaked on LB solid medium containing 50. Mu.g/mL kanamycin, and cultured at 37 ℃ for 12 hours. A single colony is picked up and is subjected to amplification culture in an LB liquid culture medium containing 50 mu g/mL kanamycin, and when the OD value of the culture solution at 600nm is about 0.6, 1mM IPTG is added to the culture solution to perform induction expression for 18 to 20 hours at the temperature of 20 ℃.

(3) Purifying by column chromatography

S1, centrifuging 3500g of fermentation liquor induced and expressed in the step (2) at 4 ℃ for 30min, taking the precipitate, redissolving the precipitate by using a binding buffer solution (50mM Tris,300mM NaCl, pH8.0), crushing the precipitate at 700bar under high pressure for 5min, centrifuging 8000g of the precipitate at 4 ℃ for 30min, and taking the supernatant.

S2, filling a gravity column with Ni NTA filler (Tiandi people and Changzhou) for nickel affinity purification, and specifically comprising the following steps:

adding the supernatant obtained in the step S1 into a gravity column, and collecting effluent liquid (penetrating fluid); washing with 10 times column volume of binding buffer solution to obtain washing solution; washing impurities by using a washing liquid with 10 times of column volume to obtain an impurity washing liquid; eluting with 3 times of eluent to obtain eluent, removing imidazole from the eluent in binding buffer solution to obtain recombinant Portunus trituberculatus rSCP, sequencing to obtain Portunus trituberculatus rSCP with sequence shown in SEQ ID NO. 2 and expression effect shown in FIG. 2.

Wherein the cleaning solution is 50mM Tris,300mM NaCl,20mM imidazole, pH8.0; the eluent was 50mM Tris,300mM NaCl,500mM imidazole, pH8.0.

As can be seen from figure 2, the high-purity Portunus trituberculatus rSCP is obtained after column chromatography purification. The purity of portunus trituberculatus rSCP in the eluate was calculated to be >97% using Image J.

Example 3

IgE reaction of Portunus trituberculatus SCP and serum of allergic patients

The IgE reaction experiment was performed by selecting 5 allergic patients with serum numbers 1-5 as the experimental group and 2 healthy patients with serum numbers 6 and 7 as the control group.

1. ELISA (enzyme-Linked immuno sorbent assay) for detecting IgE binding capacity of portunus trituberculatus nSCP

The indirect ELISA experiment comprises the following specific steps:

coating: nSCP was diluted to 10. Mu.g/mL using CBS solution (0.05M, pH 9.6), 100. Mu.L/well coated for 2h at 37 ℃. The plate solution was spun off and washed 5 times with 300. Mu.L/well PBST.

And (3) sealing: add 150. Mu.L of 1% BSA in PBST per well and block for 2h at 37 ℃. The plate solution was spun down and washed 5 times with 300. Mu.L/well PBST.

Primary antibody incubation: add 100. Mu.L of serum or PBST to each well, set 3 replicates, and incubate for 1.5h at 37 ℃. The plate solution was spun off and washed 5 times with 300. Mu.L/well PBST.

And (3) secondary antibody incubation: mu.L of HRP-goat anti-human IgE secondary antibody diluted 1:2000 was added to each well and incubated at 37 ℃ for 1h. The plate solution was spun down and washed 5 times with 300. Mu.L/well PBST.

Color development: 100 mu L of TMB single-component color developing solution (Solebao, beijing) is added into each hole, color development is carried out for 10min at 37 ℃,50 mu L/hole stop solution (2M sulfuric acid) is immediately added to stop reaction, and the light absorption value is measured at 450 nm.

The result is shown in A in figure 3, the OD values of the serum of the allergic patient are very different from those of the healthy serum, and the blue crab nSCP has stronger IgE binding capacity with the serum of the allergic patient.

2. Dot blot for detecting IgE binding capacity of portunus trituberculatus nSCP

The Dot blot experiment comprises the following specific steps:

coating: after the NC membrane was wetted with ultrapure water, ncps 2 μ g was spotted therein.

And (3) sealing: blocking with 5% BSA,3 times PBST washing.

Primary antibody incubation: mu.L of the above human serum was spotted on NC membranes, incubated overnight at 4 ℃ and washed 3 times with PBST.

And (3) secondary antibody incubation: HRP-sheep anti-human IgE 1 dilution, incubation for 4h, PBST washing 3 times.

Color development: images were developed using ELC two-component developing solution (Thermo Fisher Scientific, USA) and photographed by chemical development using a gel imager (Bio-Rad, USA).

As a result, as shown in B in FIG. 3, the serum of the allergic patients had a significant difference from the serum of the healthy patients with visual observation.

The results in figure 3 show that the portunus trituberculatus nSCP has stronger IgE binding capacity with the serum of allergic patients, namely the portunus trituberculatus nSCP can be used as an allergen for detection and diagnosis.

3. ELISA (enzyme-Linked immuno sorbent assay) for detecting IgE binding capacity of rSCP (ribonucleic acid) of portunus trituberculatus

The step of detecting the binding capacity of the rSCP of the blue crab and the IgE by adopting indirect ELISA is the same as the step of detecting the binding capacity of the nSCP of the blue crab by adopting ELISA except that the rSCP of the blue crab is adopted for coating in the coating step.

The result is shown in figure 4, the OD values of the serum (1-5) of the allergic patients and the serum (6-7) of the healthy patients have extremely obvious difference, the rSCP has stronger IgE binding capacity with the serum of the allergic patients, and the rSCP of the blue crab can be used as an allergen for detection and diagnosis.

Example 4

The detection method of the double-antibody sandwich ELISA comprises the following steps:

coating: rat anti-rscpig polyclonal antiserum was diluted with CBS at 1 10000, 100 μ L/well, overnight at 4 ℃, and washed 3 times with PBST. Wherein, the polyclonal antibody is prepared by adopting a common method for immunization.

And (3) sealing: BSA 1% 200. Mu.L/well, blocking at 37 ℃ for 2h, PBST washing 3 times.

Antigen: rSCP was diluted to different concentrations, 100. Mu.L/well, incubated 40min at 37 ℃ and PBST washed 5 times.

A first antibody: rabbit anti-rSCP IgG polyclonal antiserum was diluted 1 20000-fold, 100 μ L/well, incubated 40min at 37 ℃, and pbst washed 5 times.

Enzyme-labeled secondary antibody: HRP-goat anti-rabbit IgG, 1.

Color development: TMB monocomponent color developing solution 100. Mu.L/well, and incubation at 37 ℃ for 20min.

Termination and measurement: 50 μ L/well stop solution, read OD 450nm minus background at 630 nm.

A standard curve is established by adopting a four-parameter fitting method, and the rSCP concentrations are respectively 1, 10, 100, 200, 400, 600, 800 and 1000ng/mL. The detection limit LOD = (average of 10 blank values +3 times standard deviation) corresponding concentration. Limit of quantitation LOQ = (mean of 10 blanks +10 standard deviations) corresponding concentration.

As a result, as shown in FIG. 5, the limit of detection LOD was 0.29ng/mL, and the limit of quantitation LOQ was 0.77ng/mL.

Example 5

Evaluation of specificity of Sandwich ELISA detection method

Selecting different foods (Eriocheir sinensis, humulus Grosvenorii, litopenaeus vannamei, arrax Armata, mantis oralis, os Sepiae, haliotis discus hannai, chlamys farreri, pacific pollack, japanese sea bass, chicken, and cattle), and extracting crude protein from edible muscle part with PBST. The crude extract was adjusted to the same protein concentration and assayed using the ELISA sandwich assay described in example 4. The concentration of the portunus trituberculatus is measured to be C according to a standard curve formula ₀ Other concentration is C _n Cross reaction rate = C _n /C ₀ ×100％。

As shown in FIG. 6, the cross-reactivity rate was low, so that the specificity of ELISA detection using blue crab SCP according to the present invention was strong.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims (10)

1. The application of the blue crab nSCP or rSCP in the preparation of the reagent for diagnosing the allergic diseases is characterized in that the nucleotide sequence of the nSCP is shown in SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown in SEQ ID NO. 2.

2. The use according to claim 1, wherein the allergic disease is caused by blue crab.

3. An application of blue crab nSCP or rSCP in preparing a food allergen detection reagent is characterized in that the nucleotide sequence of the nSCP is shown in SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown in SEQ ID NO. 2.

4. An ELISA kit for detecting Portunus trituberculatus SCP allergen is characterized in that the kit comprises Portunus trituberculatus SCP; the SCP comprises nSCP or rSCP, wherein the nucleotide sequence of nSCP is shown in SEQ ID NO. 1, and the nucleotide sequence of rSCP is shown in SEQ ID NO. 2.

5. The ELISA kit of claim 4 further comprising a coating diluent, a blocking solution, a washing solution, a developing solution, a stop solution and a negative quality control.

6. The ELISA kit of claim 5 wherein the negative quality control is human serum.

7. The use according to any of claims 1 to 3, wherein the preparation method of said rSCP comprises the following steps:

the His tag sequence, the enterokinase site sequence and the Portunus trituberculatus nSCP sequence are inserted into a pET-30a plasmid to construct a recombinant plasmid, and then the recombinant plasmid is transformed into a competent cell and is induced to express.

8. The use of claim 7, wherein the His tag sequence is shown as SEQ ID NO. 3, the enterokinase site sequence is shown as SEQ ID NO. 4, and the Portunus trituberculatus nSCP sequence is shown as SEQ ID NO. 1.

9. The use of claim 7, wherein the preparation of rSCP further comprises column chromatography purification, wherein the column chromatography purification comprises:

centrifuging and crushing fermentation liquor for induced expression, taking supernatant, adding the supernatant into a gravity column for nickel affinity purification, and washing by using a binding buffer solution, a cleaning solution and an eluent in sequence to obtain rSCP.

10. The use of claim 9, wherein the binding buffer comprises 45-55mM tris, 280-320 mM NaCl;

the cleaning solution comprises 45-55mM Tris, 280-320mM NaCl and 15-25 mM imidazole;

the eluent comprises 45-55mM Tris, 280-320mM NaCl and 490-510 mM imidazole.

Images