CN115963273B - Application and kit of portunus trituberculatus nSCP or rSCP - Google Patents
Application and kit of portunus trituberculatus nSCP or rSCP Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides an application and a kit of a portunus trituberculatus nSCP or rSCP, belonging to the technical field of allergen detection, wherein the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, the nucleotide sequence of the rSCP is shown as SEQ ID NO. 2, the 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 detection limit is 0.29ng/mL, the quantitative limit is 0.77ng/mL, and the cross reaction rate is low when the detection of the portunus trituberculatus troponin is utilized, so that the invention enriches the types of deficient portunus trituberculatus allergens, can improve the diagnosis and immunotherapy level related to crab allergic diseases, and can improve the detection accuracy and specificity of crab allergens in foods.
Description
Technical Field
The invention belongs to the technical field of allergen detection, and particularly relates to application and a kit of portunus trituberculatus nSCP or rSCP.
Background
Crustaceans are one of the eight major classes of major allergic foods proposed by the united kingdom grain and farming organizations and the world health organization, wherein crab allergy is one of the most common causes of food allergy in asian areas such as china, japan, thailand, etc. In view of the economic body quantity and allergic incidence of the portunus trituberculatus, the method has important economic and clinical significance for the research of the allergen of the portunus trituberculatus.
However, the current research on the allergens of portunus trituberculatus is quite deficient. The world health organization (World Health Organization, WHO) and International immunology Association (International Union of Immunological Societies, IUIS) authorized allergen nomenclature Commission sites (http:// www.allergen.org /) have not been provided with any Portunus trituberculatus allergen proteins. In view of the fact that the sequence types of the portunus trituberculatus allergen are not comprehensive, the properties such as sensitization are not clear, so that a reasonable detection standard is lacking for accurately detecting food allergens at present, and a comprehensive and proper diagnosis preparation is lacking for accurately diagnosing allergic patients. Therefore, the system identification and research of the portunus trituberculatus allergen are carried out, each allergen protein is known, the properties of the allergen protein are utilized to carry out the combination of a plurality of allergens or the application of single components, and the stable and accurate detection and diagnosis result are obtained.
The troponin (Sarcoplasmic calcium binding protein, SCP) has the characteristics of high heat and acid-base stability and strong species specificity, and has potential for allergen detection and diagnosis. However, no research on SCP allergy of portunus trituberculatus is currently seen. Due to the sequence differences of SCPs among species, epitopes thereof are different. Therefore, the SCP sequences of other species cannot replace the SCP of the portunus trituberculatus for food detection or allergy diagnosis, because the wrong detection and diagnosis are likely to be caused or the allergy condition of the patient to the portunus trituberculatus cannot be clarified, and extra economic burden and life pressure of the patient are caused.
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 problem that the allergen of portunus trituberculatus in food cannot be accurately and specifically detected and the allergic diseases caused by the portunus trituberculatus cannot be accurately diagnosed.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of a portunus trituberculatus nSCP or rSCP in preparing a reagent for diagnosing allergic diseases, wherein the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown as SEQ ID NO. 2.
Preferably, the allergic disease is caused by portunus trituberculatus.
The invention provides an application of portunus trituberculatus nSCP or rSCP in preparing a food allergen detection reagent, wherein the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown as SEQ ID NO. 2.
The invention provides an ELISA kit for detecting SCP allergen of portunus trituberculatus, which comprises SCP of portunus trituberculatus; the SCP comprises nSCP or rSCP, wherein the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown as SEQ ID NO. 2.
Preferably, the kit further comprises coating diluent, sealing liquid, washing liquid, color development liquid, termination liquid and negative quality control.
Preferably, the negative quality control is serum of a healthy person.
Preferably, the preparation method of the rSCP comprises the following steps:
his tag sequence, enterokinase site sequence and Portunus trituberculatus nSCP sequence are inserted into pET-30a plasmid to construct recombinant plasmid, and then the recombinant plasmid is transformed into competent cells, and induced expression is performed.
Preferably, 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.
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 inducing expression, taking 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 45-55 mM Tris, 280-320 mM NaCl;
the cleaning solution comprises 45-55 mM Tris, 280-320 mM NaCl and 15-25 mM imidazole;
the eluent comprises 45-55 mM Tris, 280-320 mM NaCl and 490-510 mM imidazole.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for detecting the serum of the allergic patient by using the mythic calcium binding protein in the portunus trituberculatus as an allergen for the first time, wherein the portunus trituberculatus nSCP or rSCP has stronger IgE binding capacity with the serum of the allergic patient. The detection limit is 0.29ng/mL, the quantitative limit is 0.77ng/mL, and the cross reaction rate is low when the detection of the portunus trituberculatus troponin is utilized, so that the invention enriches the types of deficient portunus trituberculatus allergens, can improve the diagnosis and immunotherapy level related to crab allergic diseases, and can improve the detection accuracy and specificity of crab allergens in foods.
Drawings
FIG. 1 is an agarose gel electrophoresis diagram of PCR amplification products of a portunus trituberculatus SCP CDS gene; in the figure: 1. DL 2000Plus DNA Marker,2, PCR amplification products;
FIG. 2 is a diagram showing the recombinant expression effect of portunus trituberculatus rSCP; in the figure: 1. cell disruption supernatant, 2, cell disruption precipitation, 3, penetrating fluid, 4, washing fluid, 5, impurity washing fluid, 6, eluent;
FIG. 3 is a graph showing the IgE response of Portunus trituberculatus nSCP to serum from allergic patients; in the figure: A. an indirect ELISA result diagram, B, dot Blot result diagram;
FIG. 4 is a graph showing the IgE response results of Portunus trituberculatus rSCP with serum from allergic patients;
FIG. 5 is a standard curve of SCP sandwich ELISA of Portunus trituberculatus;
FIG. 6 shows the results of specific evaluation of sandwich ELISA.
Detailed Description
The invention provides an application of a portunus trituberculatus nSCP or rSCP in preparing a reagent for diagnosing allergic diseases, wherein the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown as SEQ ID NO. 2.
In the invention, the portunus trituberculatus myogenic calcium binding protein disclosed by the invention is higher in homology of the n SCP of the portunus trituberculatus and other crab SCP sequences. The feasibility of the SCP of the portunus trituberculatus in terms of allergen and detection and diagnosis is verified by indirect ELISA or Dot Blot between the nSCP or rSCP of the portunus trituberculatus and serum of an allergic patient. The allergic disease is preferably caused by portunus trituberculatus.
The invention provides an application of portunus trituberculatus nSCP or rSCP in preparing a food allergen detection reagent, wherein the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of the 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 paste, crab meat, crab sticks, crab cream noodles, crab cream shortbread, crab cream crust, and the like.
The invention provides an ELISA kit for detecting SCP allergen of portunus trituberculatus, which comprises SCP of portunus trituberculatus; the SCP comprises nSCP or rSCP, wherein the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown as SEQ ID NO. 2.
In the invention, the portunus trituberculatus myotonin is named as portunus trituberculatus nSCP, and the recombinant portunus trituberculatus myotonin is named as portunus trituberculatus rSCP. The nucleotide sequence of the nSCP is as follows:
atggcttactcttgggacaaccgcgtcaagtatgtcgtcaggtacatgtacgacatcgacaacaatggctacctcgacaagaatgactttgagtgcctggcgctgaggaacacactcatcgagggccgcggcgaattcaactccgacgcctatgccaacaaccagaagattatgagcaacctctggaacgagatcgccgagctggctgatttcaacaaggacggtcaggttactgtggacgagttcaaacaagccgtccagaagctgtgttgcggcaagaactttgatgctttccctccttgcttcaagactgtcattggtcacctgttcaagactattgatatcaacggtgacggccttgttggcgttgatgagtacaggctggattgcatctccaggagtgccttctcctccgttaaggagattgatgatgcttatggcaagctctgcactgacgatgacaagaaggctggtggcatcagcctcagccgttaccaggagctgtatgcccagttcatctccaaccctgacgagaagtgcaatgccgtctaccttttcggacccctgaaggaggtgcagtaa。
in the present invention, the nucleotide sequence of the rSCP is as follows:
atgcaccatcatcatcatcatgacgacgacgacaagatggcatatagctgggataaccgtgttaaatatgttgttcgttatatgtatgatatcgataacaacggttatctggataaaaacgattttgaatgtctggccctgcgtaatacgctgattgaaggtcgcggtgaatttaatagtgatgcatacgctaataaccagaaaattatgagtaacctgtggaatgaaatcgcagaactggccgattttaacaaagatggtcaggttaccgttgatgaatttaaacaggccgtgcagaaactgtgttgtggcaaaaattttgatgcgtttccgccttgttttaaaaccgttattggtcatctgtttaaaaccattgatatcaatggcgatggtctggttggcgttgatgaatatcgtctggattgtattagtcgtagtgcattttctagtgttaaagaaattgatgatgcctatggtaaactgtgtaccgatgatgataaaaaggcgggtggtattagtctgagtcgttatcaggaactgtatgcccagtttattagtaacccggatgaaaaatgtaacgcagtttatctgtttggtcctctgaaagaagtacagtaa。
in the invention, the kit further comprises coating diluent, sealing liquid, washing liquid, color development liquid, termination liquid and negative quality control. The coating diluent is preferably a CBS solution. The blocking solution is preferably PBST containing 0.5% -6% BSA. The wash solution is preferably PBST. The color developing solution is preferably TMB single-component color developing solution. The stop solution is preferably 1.5-2.5M sulfuric acid. The negative quality control is preferably serum of a healthy person. The source of the CBS solution is not particularly limited, and the CBS solution is prepared by using products sold in the field or a conventional preparation method.
In the present invention, the preparation method of the rSCP preferably includes the following steps:
his tag sequence, enterokinase site sequence and Portunus trituberculatus nSCP sequence are inserted into pET-30a plasmid to construct recombinant plasmid, and then the recombinant plasmid is transformed into competent cells, and induced expression is performed.
In the invention, the His tag has little influence on the structure and the biological activity of the portunus trituberculatus rSCP, and the protein purification of the portunus trituberculatus rSCP is utilized. The His tag sequence is preferably shown in SEQ ID NO. 3, wherein the His tag sequence is caccatcatcatcatcat. In the invention, enterokinase has the characteristics of high specificity and high hydrolysis efficiency, and can be used as protease to specifically identify and cut a substrate containing an enterokinase cutting site, wherein the enterokinase site sequence is preferably shown as SEQ ID NO. 4, and the enterokinase site sequence is gacgacgacgacaag; the sequence of the Portunus trituberculatus nSCP is preferably shown as SEQ ID NO. 1. In the present invention, if rSCP completely consistent with nSCP sequence is to be obtained, a common enterokinase product in the market is selected, and His tag sequence and enterokinase site are excised according to the commodity instruction manual.
As a preferred embodiment, the method for preparing the rcpp further comprises column chromatography purification, and the method for column chromatography purification comprises:
centrifuging and crushing the fermentation liquor for inducing expression, taking 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.
In the present invention, the binding buffer is a buffer system in which rSCP is bound to the filler, and preferably the binding buffer includes 45 to 55mM Tris,280 to 320mM NaCl. The washing solution of the present invention, which preferably comprises 45 to 55mM Tris,280 to 320mM NaCl,15 to 25mM imidazole, uses low concentration imidazole to remove poorly bound proteins. The eluent of the present invention uses high concentration imidazole to compete for eluting target protein, and the eluent preferably comprises 45-55 mM Tris, 280-320 mM NaCl and 490-510 mM imidazole. The purity of rSCP in the eluent is calculated by using Image J to be more than 97%, so that the purity of the rSCP protein 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 the CDS sequence of the Portunus trituberculatus troponin
(1) Total RNA extraction
Total RNA extraction of portunus trituberculatus muscle was performed according to the procedure described using a total RNA extraction kit (soribao, beijing).
(2) Reverse transcription
Immediately after RNA extraction, 5. Mu.L of RNA was digested with genomic DNA and reverse transcribed to cDNA using HiScript III RT SuperMix for qPCR (+gDNA wind) (Northenzan, nanjing) according to the protocol, and the product was stored in aliquots at-20 ℃.
(3) Primer design and Synthesis
Sequence design primers outside the CDS region of the selected transcriptome are shown in Table 1 for primer information.
TABLE 1 primer information
(4) PCR amplification
PCR amplification was performed using the cDNA obtained by the reverse transcription as a template and the F/R as a primer, and 2X Rapid Taq Master Mix (Northenan, nanjing).
PCR amplification reaction system: cDNA template 1.5. Mu.L, primers 2. Mu.L each, 2X Rapid Taq Master Mix. Mu.L, ddH 2 OμL。
PCR amplification reaction conditions: after 95℃for 5min, 35 cycles were performed at 95℃for 15sec, 56℃for 15sec, and 72℃for 5sec, and finally 72℃for 5min.
(5) Product validation
The PCR product obtained by the amplification in the step (4) was subjected to 1% agarose gel electrophoresis, and the result is shown in FIG. 1.
As shown in the results of FIG. 1, the target band at about 600bp was brightly visible at a high ratio, i.e., amplified to give the target band.
(6) Sequencing analysis
The PCR amplification product is subjected to bidirectional sequencing by using a primer F, R, forward and reverse sequencing results are compared and spliced, a sequence between ATG and TAA is selected to obtain a CDS sequence of the blue crab, and the CDS sequence is compared with a reported crab SCP sequence to confirm the SCP sequence by the homology of more than 80 percent as shown in SEQ ID NO. 1.
Wherein, the amino acid sequence of the SCP of the portunus trituberculatus is shown as SEQ ID NO:7, and the method specifically comprises the following steps:
MAYSWDNRVKYVVRYMYDIDNNGYLDKNDFECLALRNTLIEGRGEFNSDAYANNQKIMSNLWNEIAELADFNKDGQVTVDEFKQAVQKLCCGKNFDAFPPCFKTVIGHLFKTIDINGDGLVGVDEYRLDCISRSAFSSVKEIDDAYGKLCTDDDKKAGGISLSRYQELYAQFISNPDEKCNAVYLFGPLKEVQ。
example 2
A preparation method of portunus trituberculatus rSCP comprises the following steps:
(1) Recombinant plasmid construction
His tag sequence (caccatcatcatcatcat), enterokinase site (gacgacgacgacaag) and Portunus trituberculatus SCP CDS sequence (SEQ ID NO: 1) are inserted between NdeI and XhoI of pET-30a plasmid enzyme cleavage site by double enzyme cleavage to construct recombinant plasmid (Optimus prime, beijing).
(2) Transformation and expression
The recombinant plasmid was transferred into competent cells E.coli BL21 (DE 3) (Bio, shanghai) by chemical transformation according to the product instructions, single colonies were picked and cultured in LB liquid medium containing 50. Mu.g/mL kanamycin at 37℃for 12h. And (3) preserving the bacterial liquid, carrying out bidirectional sequencing, and carrying out subsequent expression if the bacterial liquid 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. Single colonies are picked and cultured in LB liquid medium containing 50 mug/mL kanamycin in an expanding mode, when the OD value of the culture solution at 600nm is about 0.6, IPTG with the final concentration of 1mM is added to induce expression for 18-20 h at 20 ℃.
(3) Column chromatography purification
S1, centrifuging 3500g of the fermentation broth induced and expressed in the step (2) at 4 ℃ for 30min, taking precipitate, re-dissolving 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 the precipitate at 8000g at 4 ℃ for 30min, and taking supernatant.
S2, filling a gravity column with Ni NTA filler (Tiandi Kong, changzhou) for nickel affinity purification, wherein the specific steps are as follows:
adding the supernatant obtained in the step S1 into a gravity column, and collecting effluent (penetrating fluid); washing with 10 times of column volume of binding buffer solution to obtain washing solution; washing impurities by using a cleaning solution with a volume of 10 times of the column volume to obtain a impurity-washing solution; eluting with 3 times of eluent to obtain eluent, removing imidazole from the eluent in binding buffer solution to obtain recombinant Portunus trituberculatus rSCP, and sequencing to obtain Portunus trituberculatus rSCP with sequence shown in SEQ ID NO. 2 and expression effect shown in figure 2.
Wherein the cleaning solution is 50mM Tris,300mMNaCl,20mM imidazole, and the pH value is 8.0; the eluent was 50mM Tris,300mMNaCl,500mM imidazole, pH8.0.
As can be seen from FIG. 2, after column chromatography purification, high purity Portunus trituberculatus rSCP was obtained. 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 with serum of allergic patient
The IgE reaction experiments were performed by selecting 5 allergic patients with serum numbers 1-5 as the experimental group and 2 healthy persons with serum numbers 6 and 7 as the control group.
1. ELISA (enzyme Linked immunosorbent assay) for detecting IgE binding capacity of Portunus trituberculatus nSCP
The indirect ELISA test comprises the following specific steps:
coating: the nSCP was diluted to 10. Mu.g/mL with CBS solution (0.05M, pH 9.6) and coated at 37℃for 2h at 100. Mu.L/well. The plate was washed 5 times with 300. Mu.L/well PBST.
Closing: mu.L of PBST containing 1% BSA was added to each well and blocked at 37℃for 2 hours. The plate was washed 5 times with 300. Mu.L/well PBST.
Incubation resistance: 100. Mu.L of serum or PBST was added to each well, 3 replicates were set and incubated at 37℃for 1.5h. The plate was washed 5 times with 300. Mu.L/well PBST.
Secondary antibody incubation: 100 μl of 1:2000 diluted HRP-sheep anti-human IgE secondary antibody was added to each well and incubated at 37deg.C for 1h. The plate was washed 5 times with 300. Mu.L/well PBST.
Color development: 100. Mu.L of TMB single-component color development solution (Soy Bao, beijing) was added to each well, developed at 37℃for 10min, and the reaction was stopped by immediately adding 50. Mu.L/Kong Zhongzhi solution (2M sulfuric acid), and the absorbance at 450nm was measured.
The results are shown as A in FIG. 3, the OD values of the serum of the allergic patient and the healthy serum are extremely different, and the blue crab nSCP has stronger IgE binding capacity with the serum of the allergic patient.
2. Dot blot detection of IgE binding capacity of Portunus trituberculatus nSCP
The Dot blot experiment specifically comprises the following steps:
coating: the NC membrane was soaked with ultrapure water and then spotted with nSCP 2. Mu.g.
Closing: blocking with 5% BSA was performed for 2h and PBST washed 3 times.
Incubation resistance: the human serum 3. Mu.L was spotted on NC membrane, incubated overnight at 4℃and washed 3 times with PBST.
Secondary antibody incubation: HRP-sheep anti-human IgE 1:2000 dilution, 4h incubation, PBST wash 3 times.
Color development: the images were photographed using a gel imager (Bio-Rad, USA) using ELC two-component color development solution (Thermo Fisher Scientific, USA).
The results are shown in FIG. 3B, where the serum of allergic patients was visually distinct from healthy serum.
The results in fig. 3 show that the portunus trituberculatus nSCP has strong IgE binding capacity with serum of allergic patients, namely, the portunus trituberculatus nSCP can be used as an allergen for detection and diagnosis.
3. ELISA (enzyme-Linked immunosorbent assay) for detecting IgE binding capacity of portunus trituberculatus rSCP (r-SCP)
The procedure for detecting the IgE binding capacity of the portunus trituberculatus rSCP by using the indirect ELISA is the same as the procedure for detecting the IgE binding capacity of the portunus trituberculatus nSCP by using the indirect ELISA except that the portunus trituberculatus rSCP is used for coating in the coating step.
As shown in FIG. 4, the OD values of the serum (1-5) of the allergic patient and the OD values of the serum (6-7) of the healthy patient are extremely different, rSCP and the serum of the allergic patient have stronger IgE binding capacity, and the rSCP of the portunus trituberculatus 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-rSCPIgG polyclonal antisera were diluted 1:10000 using CBS, 100. Mu.L/well, overnight at 4℃and PBST washed 3 times. Among them, polyclonal antibodies are prepared by immunization using a general method.
Closing: 200. Mu.L/well 1% BSA, blocked at 37℃for 2h, and washed 3 times with PBST.
Antigen: rSCP was diluted to different concentrations, 100. Mu.L/well, incubated at 37℃for 40min, and PBST washed 5 times.
An antibody: the rabbit anti-rSCP IgG polyclonal antiserum was diluted 1:20000 times, 100. Mu.L/well, incubated at 37℃for 40min, and PBST washed 5 times.
Enzyme-labeled secondary antibody: HRP-goat anti-rabbit IgG,1:10000 dilution, 100. Mu.L/well, 40min incubation at 37℃and PBST wash 5 times.
Color development: TMB single-component color development solution 100. Mu.L/well, incubated at 37℃for 20min.
Termination and measurement: 50. Mu.L/Kong Zhongzhi solution, the OD at 450nm was read minus the background at 630 nm.
A standard curve is established by adopting a four-parameter fitting method, and the concentration of rSCP is respectively 1, 10, 100, 200, 400, 600, 800 and 1000ng/mL. Detection limit lod= (average value of 10 blank values+3 times standard deviation) corresponding concentration. Quantification limit loq= (average of 10 blank values +10 standard deviations) corresponding concentration.
As a result, the detection limit LOD was 0.29ng/mL, and the quantitative limit LOQ was 0.77ng/mL, as shown in FIG. 5.
Example 5
Specificity assessment of sandwich ELISA detection method
Selecting different foods (Eriocheir sinensis, litopenaeus vannamei, protoxeris, kochianus, octopus ocellatus, haliotis discus, chlamys farreri, pacific cod, japanese sea bass, chicken, and cattle), and extracting crude protein from edible muscle part by using 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-reactivity = C n /C 0 ×100％。
As shown in FIG. 6, the cross reaction rate is low, so that the specificity of ELISA detection by utilizing the SCP of the portunus trituberculatus is high.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The application of the portunus trituberculatus nSCP or rSCP in preparing the reagent for diagnosing the allergic diseases is characterized in that the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein the allergic disease is caused by portunus trituberculatus.
3. The application of the portunus trituberculatus nSCP or rSCP in preparing a food allergen detection reagent is characterized in that the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown as SEQ ID NO. 2.
4. An ELISA kit for detecting SCP allergen of portunus trituberculatus, which is characterized in that the kit comprises SCP of portunus trituberculatus; the SCP comprises nSCP or rSCP, wherein the nucleotide sequence of the nSCP is shown as SEQ ID NO. 1, and the nucleotide sequence of the rSCP is shown as SEQ ID NO. 2.
5. The ELISA kit of claim 4, wherein the kit further comprises a coating diluent, a blocking solution, a washing solution, a color development solution, a stop solution and a negative quality control.
6. The ELISA kit of claim 5, wherein the negative quality control is healthy human serum.
7. The use according to any one of claims 1 to 3, wherein the preparation method of the rSCP comprises the steps of:
his tag sequence, enterokinase site sequence and Portunus trituberculatus nSCP sequence are inserted into pET-30a plasmid to construct recombinant plasmid, and then the recombinant plasmid is transformed into competent cells, and induced expression is performed.
8. The use according to claim 7, wherein the His tag sequence is shown in SEQ ID No. 3, the enterokinase site sequence is shown in SEQ ID No. 4, and the portunus trituberculatus ncp sequence is shown in SEQ ID No. 1.
9. The use of claim 7, wherein the method of preparing the rSCP further comprises column chromatography purification, the method comprising:
centrifuging and crushing the fermentation liquor for inducing expression, taking 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.
10. The use according to claim 9, wherein the binding buffer comprises 45-55 mM tris, 280-320 mM NaCl;
the cleaning solution comprises 45-55 mM Tris, 280-320 mM NaCl and 15-25 mM imidazole;
the eluent comprises 45-55 mM Tris, 280-320 mM NaCl and 490-510 mM imidazole.
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| CN114539377A (en) * | 2022-02-25 | 2022-05-27 | 青岛海关技术中心 | Method for detecting decapod aquatic product allergen by using liquid chromatography-mass spectrometry |
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| KR20200002888A (en) * | 2017-04-28 | 2020-01-08 | 호유 가부시키가이샤 | Allergens and their epitopes |
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| CN114539377A (en) * | 2022-02-25 | 2022-05-27 | 青岛海关技术中心 | Method for detecting decapod aquatic product allergen by using liquid chromatography-mass spectrometry |
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| A multiplex PCR assay combined with capillary electrophoresis for the simultaneous detection of tropomyosin allergens from oyster, mussel, abalone, and clam mollusk species;Seung-Man Suh;Food Chemistry;20200219;第317卷;全文 * |
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