CN113416718B

CN113416718B - Specific antigen for detecting early trichinosis and application thereof

Info

Publication number: CN113416718B
Application number: CN202110578507.5A
Authority: CN
Inventors: 杨娜; 桑晓宇; 王彦虎; 丁莹莹; 冯颖; 陈冉
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2021-05-26
Filing date: 2021-05-26
Publication date: 2024-01-26
Anticipated expiration: 2041-05-26
Also published as: CN113416718A

Abstract

The invention discloses a specific antigen for detecting early trichinosis and application thereof, and the specific antigen for detecting early trichinosis comprises a first protein and a second protein, wherein the first protein is a protein of the following (a 1) or (a 2): (a 1) a protein with an amino acid sequence shown as SEQ ID.1; (a2) And (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence of the protein defined in the (a 1) and can be specifically combined with the sheep trichinosis serum antibody. The invention is used for detecting the specific antigen of the early trichinosis and the application thereof, and the detection time is advanced to 48h for the first time, so that the operation is simple and convenient, and the sensitivity and the accuracy are good.

Description

Specific antigen for detecting early trichinosis and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a specific antigen for detecting early trichinosis and application thereof.

Background

Trichinosis is a serious global zoonotic disease, and people or ruminants mainly attack by eating meat infected by trichina by mistake, and the disease is a second-class animal epidemic disease, which causes huge economic loss to animal husbandry every year. Currently, regarding the diagnosis of trichina, the methods accepted by the international veterinary agency (OIE) are a tablet microscopic examination method and a digestion method, however, when the two methods are detected, a certain hold-down exists, and the microscopic examination method is time-consuming, laborious and low in sensitivity. Although the digestion method can improve the detection rate of the trichina to a certain extent, the method has the advantages that the detection rate of the trichina is more than 3 trichina per gram of meat, and the method is cumbersome in operation and consumes a great amount of manpower and material resources. Immunological diagnosis, such as ELISA kit method, colloidal gold, etc., but clinical application shows that these methods have low detection accuracy and convenience, and are not convenient for basic workers to operate and detect. When animals are infected with trichina, the traditional diagnosis method is diagnosis at slaughter, however, the typical white needle tip size of the encapsulated tissue is not easy to diagnose in the slaughter process; serological diagnosis includes colloidal gold immunochromatography and enzyme-linked immunosorbent assay. These methods typically use excreted secretions as antigens, are highly false positives, are expensive to prepare and are difficult to commercialize.

In summary, the existing diagnostic means for the trichina have the defects of complicated detection process, high false positive and high detection cost, so how to improve the immunodiagnosis of the trichina and search for a reliable antigen, and establish a diagnosis technology with high efficiency, accuracy, good specificity and simplicity is a technical problem to be solved urgently.

Disclosure of Invention

For this purpose, the invention provides specific antigens for the detection of early trichinosis and uses thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides a protein for detecting early trichinosis, comprising a first protein and a second protein, the first protein being a protein of the following (a 1) or (a 2):

(a1) A protein with an amino acid sequence shown as SEQ ID.1;

(a2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence of the protein defined in the step (a 1) and can be specifically combined with the sheep trichinosis serum antibody;

the second protein is a protein of the following (b 1) or (b 2):

(b1) A protein with an amino acid sequence shown as SEQ ID.3;

(b2) And (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence of the protein defined in the step (b 1) and can be specifically combined with the sheep trichinosis serum antibody.

Nucleic acid molecules encoding said first protein or second protein are also within the scope of the present invention.

Preferably, the nucleic acid molecule is a gene encoding the first protein or the second protein,

the gene encoding the first protein is a DNA molecule as set forth in any one of the following:

(c1) A DNA molecule with a coding sequence shown as SEQ ID NO. 2;

(c2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (c 1) and which encodes the first protein of claim 1;

(c3) A DNA molecule having more than 90% homology to the DNA molecule defined in (c 1) or (c 2) and encoding said first protein;

the gene encoding the second protein is a DNA molecule as set forth in any one of the following:

(d1) A DNA molecule with a coding sequence shown as SEQ ID NO. 4;

(d2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (d 1) and which encodes said second protein;

(d3) A DNA molecule having more than 90% homology to the DNA molecule defined in (d 1) or (d 2) and encoding the second protein of claim 1.

The invention also provides application of the protein in preparing an immunochromatographic test strip for detecting trichinosis in sheep, wherein the test strip comprises a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane and the water absorption pad are sequentially and fixedly connected to the bottom plate, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is close to one end of the binding pad, the quality control line is close to one end of the water absorption pad, and the detection line is coated with the second protein according to claim 1;

the binding pad is coated with microsphere-labeled mouse IgG antibody and the first protein of claim 1;

the quality control line is coated with goat anti-mouse IgG antibody.

Preferably, the first protein is a protein with 6 histidine added at the amino terminus of the amino acid sequence shown in seq id.1;

the second protein is a protein with 6 histidine added at the amino terminal of the amino acid sequence shown in SEQ ID.3.

Preferably, the coating concentration of the antigen protein at the detection line is 2mg/mL.

Preferably, in the binding pad, the concentration of the mouse IgG label is 1mg/mL, and the concentration of the label of the first protein is 50. Mu.g/mL.

Preferably, the goat anti-mouse IgG is coated at a concentration of 2mg/mL.

In the invention, the newborn larvae specific DNaseII-11 antigen is predicted by a functional domain to find that the protein is DNase II family protein, the homology of a plurality of proteins in the family is very high, and experiments of ko further prove that the protein plays an important role in the encapsulation formation and the apoptosis process of the trichina. In recent years, research shows that DNase II family proteins are expressed in three periods ML, NBL, md of trichina, most of the proteins are secreted proteins, and the proteins can prevent the integration of exogenous genes and participate in functions such as invasion. Other functions and meanings of the trichina family proteins are not clear at present, and further research is needed. The protein has 17B cell epitopes (critical value is 0.81), and 13T cell epitopes (critical value is 21). Has good hydrophilicity, has a signal peptide and no transmembrane, and is a secretory protein.

In the invention, the time-resolved fluorescence immunochromatography technology is utilized to combine the characteristics of immunolabeling and immunochromatography. The immunochromatography test strip is prepared by taking the time-resolved fluorescent microspheres as tracers, can be clinically operated simply, rapidly diagnosed, has high sensitivity and strong specificity, and has wide application prospect in a method for qualitatively and accurately detecting the trichina infected in the cattle and sheep.

The established method is based on double-antigen sandwich immunochromatography, and the trichina antibody in the sample is detected according to the specific reaction of the antigen and the antibody and the immunochromatography analysis technology. Fluorescent microsphere-labeled recombinant antigen and mouse IgG antibody are coated in a fluorescent binding pad (glass fiber membrane), meanwhile, recombinant antigen is coated on a detection line on a nitrocellulose membrane, and goat anti-mouse IgG antibody is coated on a quality control line. When detecting, if the sample contains the trichina IgG antibody, the IgG antibody in the sample can be combined with the fluorescent-antigen in the combining pad at the front end of the test strip, and the fluorescent-antigen-antibody-antigen immune complex is formed by capturing the other recombinant antigen in the detection area due to the siphon effect flowing along the lateral direction of the chromatographic strip, and the fluorescent intensity emitted by the rare earth ions in the complex is measured by using a fluorescent immunoassay analyzer. Under the projection of an ultraviolet lamp (365 nm), the test area and the control area of the chromatographic test strip excite the attached fluorescent microsphere conjugate, the emitted light is gathered and converted into an electric signal, the intensity of the electric signal is closely related to the number of fluorescent molecules, and the content of an analyte in a sample to be tested can be automatically analyzed by a fluorescence analyzer.

The invention has the following advantages:

the invention is used for detecting the specific antigen of the early trichinosis and the application thereof, the detection time is advanced to 48 hours for the first time, and the operation is simple and convenient, and the sensitivity and the accuracy are good;

the detection method established by the invention has good specificity, provides a rapid diagnosis method for epidemiological investigation and disease diagnosis, and can detect the serum of early-stage infected trichina at the same time.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.

The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.

FIG. 1 is a diagram showing the double restriction enzyme digestion verification of the recombinant expression plasmid of putave try psin (Tsp_00436) -pET-28a, wherein the recombinant expression plasmid of putave try psin (Tsp_00436) -pET-28a is digested by 1-NdeI and XhoI; 2-putative try-28 a recombinant expression plasmid (tsp_00436) -pET-28 a;

FIG. 2 shows SDS-PAGE and Western-blot verification of the push try protein (Tsp_00436) -His recombinant protein provided by the embodiment of the invention;

FIG. 3 is a diagram showing the double digestion verification of the recombinant expression plasmid pET-28a-newborn larvae specific DNaseII-11 provided by the embodiment of the invention, wherein the recombinant expression plasmid pET-28 a-push tree (Tsp_00436) is digested with 1-NdeI and XhoI; 2-pET-28 a-push try tree (Tsp_00436) recombinant expression plasmid;

FIG. 4 shows SDS-PAGE and Western-blot verification of newborn larvae specific DNaseII-11-His recombinant proteins provided by the embodiment of the invention;

FIG. 5 is a schematic structural diagram of a time-resolved fluorescence microsphere chromatography test strip according to an embodiment of the present invention, wherein the test strip is a 1-PVC base plate; 2-sample pad; 3-a bonding pad; 4-water absorbing paper; a 5-nitrocellulose membrane; 6-detecting line T; 7-a quality control line C;

FIG. 6 shows the result of specific detection of a time-resolved fluorescence microsphere chromatography test strip provided by the embodiment of the invention, wherein 1-sheep trichinosis positive serum; 2-sheep supine nematode positive serum; 3-ovine prandial nematode positive serum; 4-ovine trichodesmus positive serum;

fig. 7 is a graph of a time-resolved fluorescence microsphere chromatography test strip sensitivity detection result provided by the embodiment of the invention, wherein 1-4: serum was sequentially mixed in 1:400,1:800,1:1600,1:3200,5: a sample diluent;

FIG. 8 is a diagram showing the detection results of the test strip provided by the embodiment of the invention for detecting the serum and negative serum of the trichinosis in different periods.

Detailed Description

Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

The present embodiment provides a preparation method of a recombinant antigen of putative tree-shaped psin (tsp_00436), wherein the amino acid sequence of the recombinant antigen of putative tree-shaped psin (tsp_00436) after optimization of the gene sequence of putative tree-shaped psin (tsp_00436) is shown as SEQ ID NO.1, and the gene sequence encoding the protein is shown as SEQ ID NO. 2. The amino acid sequence of newborn larvae specific DNaseII-11 recombinant antigen is shown as SEQ ID NO.3, and the gene sequence encoding the protein is shown as SEQ ID NO. 4.

1. The gene fragment is synthesized by Hongsun biotechnology Co.Ltd into expression vectors putave try psin (Tsp-00436), newborne-specific DNaseII-11 containing corresponding target genes, and the steps of double enzyme digestion are as follows:

(1) Centrifuging a centrifuge tube filled with the expression vector freeze-dried powder of the target gene at 3000 rpm/normal temperature for 1min.

(2) With 50. Mu.L of sterile ddH ₂ The O-dissolved dry powder was thawed, then gently mixed with a vortex, and then immediately separated for 30s with a palm centrifuge.

(3) The concentration was measured using NANODROP.

(4) The expression vector plasmid pET-28a was digested with restriction enzymes, and the system for digestion is shown in Table 1.1. The reaction conditions were 37℃for 10min.

TABLE 1.1pET-28a expression plasmid double enzyme digestion system

2. Recombinant plasmid transformation expression competence

(1) 50. Mu.L of BL21 (DE 3) after ice bath was placed in a 1.5mL sterile centrifuge tube, melted in an ice box (rubbed without hands), and 5. Mu.L of pET-28 a-push try and pET-28a-newborn larvae specific DNaseII-11 expression plasmids were respectively added thereto by pipetting, and allowed to stand on ice for 30min.

(2) After ice bath, the centrifuge tube was placed in a water bath, floated for 45S under thermal shock at 42 ℃, and then the tube was transferred to ice for ice bath for 2min.

(3) In a sterile super clean bench, 400-500. Mu.L of sterile liquid LB medium was added to each centrifuge tube at 220rpm, and cultured in a shaker at 37℃for 1h.

(4) Firstly, placing the LB solid plate with the corresponding resistance at 37 ℃ for preheating, and taking out for standby after the water vapor in the plate is completely volatilized.

(5) According to experimental requirements, absorbing a proper amount of transformed competent cells, dripping the competent cells in a preheated plate in a scattering manner, uniformly coating the competent cells, and placing the competent cells in a 37 ℃ incubator.

(6) The cells were cultured overnight at 37℃in a constant temperature incubator.

3. Small-scale inducible expression of His-tagged recombinant proteins

(1) Single colonies of BL21 (DE 3) after transformation were picked with a sterile white tip, and after PCR identification was correct, inoculated in 10mL of liquid LB medium containing the corresponding resistance, and incubated overnight at 200rpm/37 ℃.

(2) The next day, after sterilization in a sterile operating table, 100. Mu.L of the bacterial liquid is sucked by a liquid transferer and inoculated into 10mL of liquid LB medium containing relative resistance, and cultured at 37 ℃ and 200rpm, and the bacterial liquid OD _600nm When=0.6-0.8, IPTG induction can be added.

(3) 100. Mu.L of uninduced whole bacteria were aspirated in a sterile control station prior to the addition of the protein inducer, as negative controls. Next, 0.1M IPTG inducer was added so that the final concentration of IPTG became 0.1mM, 0.2mM, 0.5mM, and induction was performed at different temperatures (16 ℃ C., 32 ℃ C., 37 ℃ C.).

(4) Collecting bacterial liquid which is cultured for 16 hours at a low temperature of 16 ℃, 8 hours at a low temperature of 32 ℃ and 5 hours at a constant temperature of 37 ℃, placing the collected bacterial liquid into a 15mL enzyme removal tube, centrifuging at 4000rpm of a centrifuge at 4 ℃ for 15min, discarding supernatant, re-suspending bacterial precipitation by using 1mL of phosphate buffer, taking 60 mu L of bacterial liquid as a whole bacterial sample after induction, crushing the rest bacterial liquid by using a small ultrasonic crusher, wherein the crushing power of the ultrasonic crusher is 28w, ultrasonic processing is carried out for 2 seconds, stopping for 3 seconds, observing bacterial clarity, placing the crushed bacterial liquid into a 1.5mL EP tube, centrifuging at 5000rpm for 10min in a centrifuge at 4 ℃, taking 60 mu L of supernatant (supernatant after induction), discarding supernatant, re-suspending precipitation by using 1mL of phosphate buffer, and taking 60 mu L of precipitate after induction.

(5) Supernatant, sediment and whole bacteria under different temperatures and different IPTG concentrations are respectively added into 20 mu L of 1 XSDS-PAGE protein loading buffer, and the mixture is boiled in boiling water at 100 ℃ for 10min and then taken out for standby.

(6) Taking 10 mu L of prepared whole bacteria, supernatant and precipitated protein samples with different temperatures and different IPTG concentrations (protein samples with different IPTG concentrations at the same temperature are sequentially loaded for subsequent observation) and loading the samples into 10% SDS-PAGE protein gel electrophoresis, wherein the setting power of a protein electrophoresis tank is as follows: the voltage is 150V and the time is 55min.

(7) Placing the cut albumin glue into a protein staining box which is poured into G250-coomassie brilliant blue staining solution in advance, and shaking the protein staining box on a horizontal shaking table for 70min.

(8) And (3) placing the dyed albumin glue into a decoloring solution, after decoloring overnight, observing the expression condition of the recombinant protein by using a glue irradiation instrument, and simultaneously determining whether the target gene transferred into the expression competence is expressed, the expression quantity and the expression form are soluble in the supernatant or inclusion bodies in the precipitate.

In this step, both proteins were induced to be expressed by inclusion bodies in small amounts, the optimal induction temperature was 37℃and the IPTG concentration of tsp_00436 was 0.2mM. The IPTG concentration of the inducible expression newborn larvae specific DNaseII-11 was 0.5mM.

4. Large scale expression and purification of His-tag inclusion body recombinant proteins

(1) Taking out bacterial liquid with His tag recombinant protein from a refrigerator at-20 ℃ in advance, putting the bacterial liquid into a refrigerator at 4 ℃, after the bacterial liquid is completely melted, picking a proper amount of bacterial liquid in an aseptic super clean bench by using an inoculating loop, rapidly scribing a Kana resistant culture plate by adopting a cross scribing method, and putting a culture dish into a constant temperature incubator at 37 ℃ for culturing for 10 hours.

(2) The next day the dishes were removed, 1-2mm single colonies were picked with white tips and inoculated into 20mL Kana resistant liquid LB medium, 200rpm in a 37℃constant temperature shaker, and cultured overnight.

(3) In a sterile operating table, 5mL of the overnight cultured bacterial liquid was aspirated, and the bacterial liquid was added to 500mL of Kana-resistant liquid LB medium, and after shaking at 37℃for 3 hours with a constant temperature shaker at 200rpm, the OD value of the bacterial liquid was measured.

(4) If the OD value of the spectrophotometer is between 0.6 and 0.8, the bacterial liquid is proved to reach the peak value of a growth curve, and the bacterial liquid is cultured according to the condition that the protein is optimized in the process of small-quantity induced expression.

(5) Separating the shaken bacterial liquid from the conical flask into a plurality of sterile 50mL centrifuge tubes, centrifuging at 4000rpm for 15min in a low-temperature centrifuge, discarding the supernatant in a waste liquid jar, adding 5mL of PBS into each tube, re-suspending the precipitate by a vortex, collecting into two sterile 50mL centrifuge tubes, centrifuging again under the same conditions, finally re-suspending and washing by adding 25mL of PBS into each tube of bacterial liquid, centrifuging at 4000rpm in a centrifuge at 4 ℃ for 20min, and discarding the supernatant.

(6) The cooler is pre-cooled at 4 ℃ for half an hour in advance, a high-pressure crusher is started, 75% alcohol, ddH2O and His Binding Buffer are used for sequentially passing through the crushing pipe after the air pressure buffering is finished, and the waste liquid is exhausted and discharged after 3 times.

(7) Adding a proper amount of PBS according to the bacterial precipitation amount, completely re-suspending the bacterial precipitation by a vortex device, pouring into a high-pressure crushing pipe, repeatedly crushing for 3-4 times, centrifuging by a centrifugal machine at the ultra-high speed of 4 ℃ at 12000rpm for 13min, and reserving the precipitate for standby.

(8) The precipitate was treated with 100. Mu.L of ddH ₂ Mixing O, adding 10mL of 6M-8M urea Binding Buffer, mixing, and roundingThe shape rotator was sensed overnight at room temperature.

(9) The next day, the sensed disrupted bacterial liquid was centrifuged at 12000rpm in a 4 ℃ centrifuge for 13min and the supernatant was retained for later use.

(10) The empty purification column is rinsed twice with 6M-8M urea His Binding Buffer, a small amount of His Binding Buffer is left, a proper amount of Ni-Agarose Resin is sucked up according to the amount of supernatant, the Ni-Agarose Resin is slowly added into the purification column, when Ni-beads are completely sunk to the bottom, a bottom switch is turned on, after waste liquid is discharged, a proper amount of His Binding Buffer filter column is used for washing the filler.

(11) Adding the supernatant collected in the step (9) into the treated Ni-Agarose Resin filler, placing the mixture on a horizontal rotator, and slowly sensing the mixture at room temperature for 3 hours.

(12) Pouring the mixture of the combined supernatant and the Ni-Agarose Resin into a purification column, opening a bottom switch after the Ni-Agarose Resin is completely precipitated, adjusting the flow rate, and finishing passing through the column when the Ni-Agarose Resin is completely in the purification column.

(13) The first time conditions for His recombinant protein purification were explored, elution was performed with imidazole from low to high concentration: 6M-8M Urea binding buffer, 10, 20, 40, 60, 80, 100, 250, 500mM, each 3mL eluting sequentially and collecting with 2mL sterile centrifuge tube, temporarily storing in ice box.

(14) And respectively preparing samples from proteins eluted by imidazole with different gradients, column passing liquid and Ni-Agarose Resin eluted in the last time, carrying out protein gel electrophoresis, observing a target strip by a protein gel analyzer, and determining the impurity washing and eluting concentration of the protein.

In this step, the optimal imidazole concentration for the Tsp_00436 protein was 250mM. The optimal elution concentration of newborn larvae specific DNaseII-11 protein is 500mM.

5. Dialysis and concentration of His-tag inclusion body recombinant proteins

(1) The SDS-PAGE protein gel was observed, the volume of the target protein was determined according to the thickness and purity of the bands, and the protein was concentrated by centrifugation at 1500rpm for 30min using a concentrator.

(2) Firstly, one end of a dialysis bag is folded into two small folds, one end of the dialysis bag is clamped by a protein dialysis clamp, target protein is slowly added along the edge of the dialysis bag, and then the other end of the dialysis bag is folded into two small folds, clamped and subjected to gradient dialysis:

a. proteins sandwiched between dialysis bags were dialyzed overnight in 3L PBS containing 6M urea and 0.5M arginine.

b. The next day, the dialyzed proteins were observed for floc, if floc, the dialysis was terminated, if no floc, the clamped target proteins were placed in 2L PBS containing 4M urea and 0.5M arginine, dialyzed for 4h, and every 1.5h, for floc in the dialysis bag.

c. If no floccules exist, the clamped protein is put into 2L PBS containing 2M urea and 0.5M arginine, dialyzed for 4 hours, and whether floccules exist in the dialysis bag is observed every 1.5 hours.

d. If there is no floc, the clamped protein is placed in 3L PBS containing 0.5M arginine and dialyzed for 4 hours, and the presence of floc in the dialysis bag is observed every 1.5 hours.

(3) The dialyzed target protein was transferred to a 2mL sterile centrifuge tube, placed in an ice box, and after measuring its concentration with Nanodrop, the band of the target protein was observed by SDS-PAGE to determine whether to concentrate the protein.

(4) The denatured target protein is dispensed into sterile 200. Mu.L centrifuge tubes and stored at-80℃preferably immediately.

6. Western-blot identification of His tag inclusion body recombinant protein

(1) SDS-PAGE running gel is referred to in step three.

(2) Cutting PVDF film with proper size according to the size of the albumin glue, activating with methanol for 1min, cutting qualitative filter paper (3 layers are one) into proper size, soaking in film transferring liquid, removing bubbles between the filter paper and the filter paper, horizontally placing the black surface of the clip below during film transferring, sequentially arranging a water absorbing net-3 layers of qualitative filter paper-albumin glue-PVDF film-layer qualitative filter paper-water absorbing net above, closing the clip after no bubbles are ensured, placing the clip into a film transferring groove, placing the black surface of the clip into the black surface of the film transferring groove, checking the transparent surface of the clip into the red surface of the film transferring groove, starting film transferring, and setting the power as follows: the current was 175mA for 86min.

(3) After film transfer, the PVDF film is put into a plastic box containing sealing liquid (5% skimmed milk) which is prepared in advance, and put into a 37 ℃ incubator, and the sealing is carried out by shaking on a shaking table for 1h.

(4) Blocking with 5% skim milk with His-tag monoclonal antibody (1:5000), and incubation overnight at 4 ℃.

(5) PBST membrane washing is carried out for 10min each time, and 4 times of washing are carried out.

(6) AP goat anti-mouse IgG 1:5, 000PBST dilution, incubation at 37℃for 60min.

(7) Repeating the step (5).

(8) And (3) a proper amount of BCIP/NET chromogenic reagent is prepared, light-resistant chromogenic is carried out for 2-3min, and the result is observed and scanned.

The recombinant expression plasmid of the target gene putative try-psin (Tsp_00436) is synthesized, the target gene is 1275bp, the target gene is identified after digestion by NdeI and XhoI restriction enzyme, and the double digestion verification result is shown in figure 1. The purified protein was confirmed by SDS-PAGE and immunoblotting to be the same as the expected protein in size, and the recombinant protein was 55kDa in size, as shown in FIG. 2.

The recombinant expression plasmid of the target gene newborn larvae specific DNaseII-11 is synthesized, the target gene is 981bp, the NdeI and XhoI restriction enzyme is used for enzyme digestion and identification, and the double enzyme digestion verification result is shown in figure 3. The purified protein was confirmed by SDS-PAGE and immunoblotting to be the same as the expected protein in size, and the recombinant protein was 37kDa in size, as shown in FIG. 4.

Example 2

As shown in fig. 5, the present embodiment provides a time-resolved fluorescence immunochromatographic test strip for trichinosis, which comprises a sample pad 2, a conjugate pad 3, a nitrocellulose membrane 5 (NC membrane, chromatographic membrane), and a water absorbing paper 4 and a PVC base plate 1. Sample pad 2, bonding pad 3, nitrocellulose membrane 5, and absorbent paper 4 on PVC base plate 1. Wherein, one end of the nitrocellulose membrane 5 is overlapped with one end of the bonding pad 3, the other end of the nitrocellulose membrane 5 is overlapped with one end of the absorbent paper 4, and one end of the sample pad 2 is overlapped with the other end of the bonding pad 3; the binding pad 3 is coated with a goat anti-bovine IgG antibody marked by fluorescent microspheres, one side of the nitrocellulose membrane 5, which is close to the binding pad, is provided with a detection line 6, the side, which is close to the absorbent paper 4, is provided with a quality control line 7,

sample adding holes are formed in the sample pad 2, a mixture of 1mg/mL of mouse IgG and 50 mug/mL of putative trypsin (Tsp_00436) recombinant antigen marked by time-resolved fluorescent microspheres is coated in the binding pad 3, newborn larvae specific DNaseII-11 recombinant antigen is coated on a detection line 6 of the nitrocellulose membrane 5, goat anti-mouse IgG is coated on a quality control line 7, and the coating concentration of the goat anti-mouse IgG is 2mg/mL.

In order to improve the accuracy of the time-resolved fluorescence immunochromatography test strip for detecting the trichinosis in sheep, the following conditions are developed:

and (3) optimizing the precondition of the time-resolved fluorescence microsphere immunochromatographic test strip: other conditions are unchanged, only one of the variables being changed at a time. The reaction conditions are as follows: the addition amount of the positive sample and the negative sample is 75 mu L (the ratio of serum to diluent is 1:2), and the reaction time is 15min. The coating parameters are 0.75 mu L/cm, and antigen or antibody coating is carried out on NC film to form a detection line (T) and a quality control line (C).

1. Determination of coating antigen

Newborn larvae specific DNaseII-11 recombinant antigen was coated at a working concentration of 1.0mg/ml,2.0mg/ml,3.0mg/ml, and C-line goat anti-mouse IgG was coated at a concentration of 2.0 mg/ml. And (5) placing the mixture into a baking oven with the temperature of 45+/-1 ℃ and the humidity of less than or equal to 35% to be baked for 16 hours. And preparing a small sample by matching the fluorescent pad and the sample pad. The value of T, C was read in a fluorescence immunoassay analyzer, and T/C was calculated to determine the optimal concentration of the coating antigen. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when negative samples were added dropwise, the T/C value was minimal.

The results are shown in Table 1.2, where N represents a negative serum sample; p represents a positive serum sample. The optimal coating amount is achieved when the concentration of the recombinant antigen newborn larvae specific DNaseII-11 is 2.0 mg/ml.

TABLE 1.2 detection results of coating antigen concentration

2. Determination of the concentration of labeled antigen

The treated fluorescent microspheres are used for respectively marking different amounts of putative try psin (Tsp_00436) antigens by the same marking method, wherein the marking amounts are respectively as follows: 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml. The samples were diluted at 4-fold dilution to prepare a small sample, matched with the coated sheet and sample pad, and the values were read T, C in a fluoroimmunoassay analyzer to calculate T/C to determine the optimal labeled antigen concentration. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when negative samples were added dropwise, the T/C value was minimal.

The results are shown in Table 2, where N represents a negative serum sample; p represents a positive serum sample. The optimal conjugate pad label concentration is when the labeled antigen, putative try psin (Tsp_00436), concentration is 50 μg/ml.

TABLE 2 detection results of labeled antigen concentration

3. Determination of dilution ratio of fluorescent labeling solution

The labeled fluorescent microspheres were diluted 2-fold, 4-fold, 8-fold, and 3 dilutions, respectively. Preparing a small sample, matching a coated sheet and a sample pad, reading T, C values in a fluorescence immunoassay analyzer, and calculating T/C to determine the optimal dilution ratio of the fluorescent marking solution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when negative samples were added dropwise, the T/C value was minimal.

The results are shown in Table 3, where N represents a negative serum sample; p represents a positive serum sample. When the dilution ratio of the fluorescent labeling solution is 4 times, the optimal dilution ratio of the fluorescent labeling solution is the optimal dilution ratio.

TABLE 3 detection results of dilution ratio of fluorescent labeling solution

4. Determination of fluorescent working fluid

Selecting proper fluorescent microsphere suspension according to the determined dilution ratio of the fluorescent labeling solution, wherein the suspension is divided into three groups: the first group is: 0.05MTris-HCl, 0.9% NaCl, 0.05% BSA, 0.05% Tween20, to ph=7.9; the second group is: 0.02m tris-HCl, 0.4% BSA, adjusted to ph=7.5; the third group is: 0.1MTris-HCl, 0.1% BSA (5 mL), 10% S9, 10% Tween20, 10% PEG12000, 3g trehalose. Preparing a small sample, matching a coated sheet and a sample pad, reading T, C values in a fluorescence immunoassay analyzer, and calculating T/C to determine the optimal fluorescence working solution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when negative samples were added dropwise, the T/C value was minimal.

The results are shown in Table 4, where N represents a negative serum sample; p represents a positive serum sample. The second set of fluorescent working fluids was found to be the best fluorescent working fluid.

TABLE 4 fluorescent working fluid detection results

5. Determination of sample pad treatment fluid

In order to make the antibody in the sample combine with the fluorescent microsphere labeled antigen in the combination pad better, three sample pad treatment solutions are prepared and divided into three groups. The first group is: phosphate buffer of pH7.4 at 0.02mol/L with 1% BSA, 0.1% Triton-100; the second group is: 0.1M Na ₂ B ₄ O ₇ ·10H ₂ O, 1% pvp, 0.2% Casein-Na, 1% Triton-X100, 1% Tetronic 1307, 0.2% NaN3, adjusted to ph=9.3; the third group is: 0.5M boric acid buffer, 1% triton x-100, 1% PVP, 2% NaCl, adjusted to ph=9.0. Preparing a small sample, matching a coated sheet and a fluorescent pad, reading T, C values in a fluorescent immunoassay analyzer, and calculating T/C to determine the optimal sample pad treatment fluid. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when a negative sample is added dropwise, the T/C value is minimum。

The results are shown in Table 5, where N represents a negative serum sample; p represents a positive serum sample. The second set of sample pad treatment fluids was found to be the best sample pad treatment fluid.

TABLE 5 sample pad treatment fluid detection results

6. Determination of reaction time

The 75 mu L sample is sucked by a pipette and added into 150 mu L sample buffer solution, and the mixture is fully mixed for 30s ^-1 In min, a pipette is used for sucking 75 mu L of mixed samples, the samples are dripped into a sample adding hole of a detection card, and after 10min,15min and 20min of reaction, the samples are respectively read. The value of T, C was read in a fluorescence immunoassay analyzer, and T/C was calculated to determine the optimal reaction time. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when negative samples were added dropwise, the T/C value was minimal.

The results are shown in Table 6, where N represents a negative serum sample; p represents a positive serum sample. The optimal reaction time of the chromatographic test strip is 15min.

TABLE 6 results of the reaction time measurements

7. Determination of sample buffer

To ensure adequate antigen-antibody reaction, three sample dilutions were explored, divided into three groups, each: a first group: pH=7.8, 0.02M Tris-HCl, 0.9% NaCl, 0.1% BSA, 0.5% Tween-20, 0.1% NaN ₃ The method comprises the steps of carrying out a first treatment on the surface of the Second group: pH=7.8, 0.05M Tris-HCl, 0.9% NaCl, 1.5% BSA, 0.01% Tween-20, 0.1% NaN ₃ The method comprises the steps of carrying out a first treatment on the surface of the Third group: PBS buffer at ph=7.8. The values of T, C were read at a fluoroimmunoassay analyzer and T/C calculated to determine the optimal sample dilution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when negative samples were added dropwise, the T/C value was minimal.

The results are shown in Table 7, where N represents a negative serum sample; p represents a positive serum sample. The second set of sample buffers is the best sample dilution.

TABLE 7 detection results of sample buffers

8. Determination of dilution ratio of sample

The sample and the sample buffer solution are mixed according to the proportion shown in the following table 8, the mixed sample is dripped into a sample adding hole of a detection card, a T, C value is read by a fluorescence immunoassay analyzer, and T/C is calculated to determine the optimal sample dilution proportion.

TABLE 8 dilution ratio of sample to sample buffer

The results are shown in Table 9, where N represents a negative serum sample; p represents a positive serum sample. The optimal dilution ratio is 1:2 (sample volume: sample buffer)

TABLE 9 detection results of sample dilutions

In the embodiment, in the time-resolved fluorescence immunochromatography test strip for early diagnosis of trichinosis, the concentration of newborn larvae specific DNaseII-11 recombinant antigen is 2.0mg/ml, the concentration of labeled antigen push try protein (Tsp_00436) is 50 mug/ml, the dilution ratio of fluorescent labeling solution is 4 times, and the fluorescent microsphere suspension is as follows: 0.02m tris-HCl, 0.4% BSA, adjusted to ph=7.5; sample pad treatment solution was 0.1M Na ₂ B ₄ O ₇ ·10H ₂ O、1％ PVP、0.2％ Casein-Na、1％ Triton-X100、1％Tetronic 1307、0.2％ NaN ₃ Adjusting to ph=9.3; optimal reaction time of chromatographic test strip15min. The sample buffer was 0.05M Tris-HCl, 0.9% NaCl, 1.5% BSA, 0.01% Tween-20, 0.1% NaN, pH=7.8 ₃ The method comprises the steps of carrying out a first treatment on the surface of the The sample dilution ratio, sample volume and sample buffer volume were 1:2.

Example 3 time resolved fluorescence microsphere chromatography test strip specificity

As shown in fig. 6, the samples of the sheep trichina positive serum, the sheep supreme nematode positive serum, the sheep esophageal nematode positive serum and the sheep trichostrongylus positive serum are detected, in addition to the positive serum of the trichina ovis, no signal is detected by the serum of other non-trichina ovis pathogens on the T line under the fluorescence immunoassay analyzer, and only one band appears on the C line, namely the negative is obtained.

Example 4 time resolved fluorescence microsphere chromatography test strip sensitivity

After positive serum is diluted by multiple ratio, the diluted samples are respectively dripped into a sample pad, and after 15min, the fluorescence intensity of a T line is observed under fluorescent immunochromatography. The results show that T, C lines can be clearly seen when the positive serum dilution ratio is 1:1600, and the brightness of the T line is very weak when the positive serum dilution ratio is 1:3200, but the brightness of the C line is still obvious, so that the detection result is proved to be effective. The results are shown in FIG. 7.

As shown in FIG. 8, the test strip prepared in example 2 was used to test the results of serum and negative serum detection at different stages of trichinosis.

While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

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Sequence listing

<110> Shenyang agricultural university

<120> specific antigen for detecting early trichinosis and application thereof

<130> GG21928942A

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 448

<212> PRT

<213> Artificial Sequence

<400> 1

Met Ile Arg Arg Leu Phe Gln Tyr Thr Ser Met Thr Phe Ala Trp Ile

1 5 10 15

Leu Leu Phe Leu Ser Ala Ala Ser Pro Ser Leu Gly Glu Phe Glu Cys

20 25 30

Gly Val Pro His Phe Lys Pro Tyr Ile Trp Lys Ser Gly Arg Ile Val

35 40 45

Gly Gly Thr Asp Val Arg Pro His Ser His Pro Trp Gln Ile Gln Leu

50 55 60

Leu Lys Ser Glu Thr Gly Gly Tyr Ser Ser Leu Cys Gly Gly Ser Leu

65 70 75 80

Val His Phe Gly Glu Pro Ser Asn Gly Thr Arg Phe Val Leu Thr Ala

85 90 95

Ala His Cys Ile Thr Thr Ser Asn Met Tyr Pro Arg Thr Ser Arg Phe

100 105 110

Thr Val Val Thr Gly Ala His Asn Ile Lys Met His Glu Lys Glu Lys

115 120 125

Lys Arg Ile Pro Ile Thr Ser Tyr Tyr Val Gln His Trp Asn Pro Val

130 135 140

Met Thr Thr Asn Asp Ile Ala Leu Leu Arg Leu Ala Glu Thr Val Tyr

145 150 155 160

Tyr Asn Lys Tyr Thr Arg Pro Val Cys Leu Pro Glu Pro Asn Glu Glu

165 170 175

Leu Thr Pro Gly Asp Ile Cys Val Val Thr Gly Trp Gly Asp Thr Thr

180 185 190

Glu Asn Gly Thr Thr Ser Asn Thr Leu Lys Gln Val Asp Val Lys Ile

195 200 205

Met Lys Lys Gly Thr Cys Ala Asn Val Arg Ser Glu Val Ile Thr Phe

210 215 220

Cys Ala Gly Ala Met Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp Ser

225 230 235 240

Gly Gly Pro Leu Ile Cys Lys Lys Asn Gly Lys Ser Val Gln Phe Gly

245 250 255

Val Val Ser Tyr Gly Thr Gly Cys Ala Arg Lys Gly Tyr Pro Gly Val

260 265 270

Tyr Ala Lys Val Pro Ser Tyr Val Thr Trp Leu Asn Lys Ala Ala Lys

275 280 285

Glu Leu Glu Asn Ser Pro Glu Gly Thr Val Lys Trp Ala Ser Lys Glu

290 295 300

Asp Ser Pro Val Asp Leu Ser Thr Thr Ser Arg Pro Thr Asn Pro Tyr

305 310 315 320

Thr Gly Ser Arg Pro Thr Ser Pro Ser Ser Gly Ser Arg Pro Thr Tyr

325 330 335

Pro Ser Ser Gly Ser Arg Pro Thr Ser Pro Ser Ser Gly Ser Arg Pro

340 345 350

Thr Tyr Pro Ser Ser Asp Gln Asp Gln His Leu His Leu Val Asp Gln

355 360 365

Asp Pro His Ile His Leu Val Asp Gln Asp Gln His Ile His Ile Leu

370 375 380

Asp Gln Asp Leu Leu Leu Lys Ser Gln Tyr Phe His His Thr Lys Asn

385 390 395 400

Ile Arg Gln Gln Phe Lys Asn Thr Leu Ile Val Tyr Gln Ala Glu Arg

405 410 415

Lys Glu Arg Ser Asn Thr Gln Ser His Arg Met Glu Leu Leu Gln Gln

420 425 430

His Ile Ile Thr Phe Leu Ser Lys Asn Ile Met Ile Asn Ser Leu Leu

435 440 445

<210> 2

<211> 1263

<212> DNA

<213> Artificial Sequence

<400> 2

tttgaatgcg gtgtgccaca ttttaaaccc tacatatgga aatctggtcg aattgttggt 60

ggaactgacg tacgaccaca ctcacatcca tggcagattc aattgttaaa gtcagaaacg 120

ggaggctaca gcagcttgtg cggtggtagt cttgttcatt tcggtgaacc ctcaaatggt 180

actcgattcg tacttaccgc cgcgcactgc ataactacta gcaatatgta tccaagaacg 240

tcaagattta cagttgtgac cggtgcccac aacatcaaaa tgcatgaaaa agaaaaaaag 300

cgcataccaa ttacttccta ttatgttcag cactggaacc ctgtgatgac aacaaacgac 360

attgcgttgc ttcgcctggc agaaactgtt tattataata aatatactag gcctgtctgt 420

ttgccagaac caaatgaaga attgactcct ggagatattt gcgttgtcac cggatggggt 480

gatacgactg aaaatggaac tacttctaat actttgaagc aagttgatgt caaaattatg 540

aagaaaggaa cttgtgcaaa tgtgagaagt gaagttatta ctttttgcgc tggagctatg 600

gagggtggta aagacagttg tcaaggtgat tctggtggcc cactgatatg caagaaaaat 660

gggaaaagtg ttcaattcgg tgtcgttagt tatggtactg gatgcgccag aaaaggttat 720

cccggagtgt atgccaaagt tccatcatat gtcacatggt taaataaagc tgcaaaagaa 780

cttgaaaatt ctcctgaagg aactgtaaaa tgggcttcaa aagaagattc gccagtcgat 840

ttatctacta catcaagacc aactaaccca tatactgggt caagaccgac atctccatct 900

agtggatcaa gacccacata tccatctagt ggatcaagac caacatctcc atctagtgga 960

tcaagaccca catatccatc tagtgatcaa gaccaacatc tccatctagt ggatcaagac 1020

ccacatatcc atctagtgga tcaagaccaa catatccata tactggatca agacctactc 1080

ctcaaaagcc agtatttcca tcataccaaa aatatccgcc agcagttcaa aaatacattg 1140

atagtttacc aagcggaacg caaggaacgc tcgaatacac agtcacacag aatggagtta 1200

ctacaacaac atattatcac ttttctaagt aaaaatatta tgattaattc actactgctc 1260

tga 1263

<210> 3

<211> 340

<212> PRT

<213> Artificial Sequence

<400> 3

Met Met Leu Thr Ile Ile Thr Val Ile Leu Ile Ser Leu Gly Ser Ser

1 5 10 15

Trp Ala Gln Gly Val Ala Thr Cys Lys Ala Asp Asp Asn Thr Asp Leu

20 25 30

Asn Trp Tyr Phe Val Tyr Lys Pro Pro Asn Ala Leu Gln Thr Lys Ile

35 40 45

Met Gln Ser Gly Gln Asn Pro Ala Trp Ala Arg Ser Ala Gln Ser Ile

50 55 60

Glu Ser Asn Asn Gly His Ser Ile Val Arg Thr Met Ala His Phe Val

65 70 75 80

Ala Glu Asn Gln Asn Ile Lys Val Leu Ala Tyr Ser Asp Asp Pro Pro

85 90 95

Asn Leu Pro Pro Arg Asn Glu Lys Ser Lys Ala Lys Gly Val Leu Leu

100 105 110

Ile Asp Asn Ser Gly Ala Asn Ala Ala Ala Trp Phe Val His Thr Val

115 120 125

Pro Lys Phe Leu Ser His Leu Gly Gly Tyr Ser Trp Pro Gln Thr Glu

130 135 140

Thr Ala Lys Gly His Ile Phe Leu Cys Leu Ser Ile Asn Glu Glu Ser

145 150 155 160

Leu Asn Ala Val Ala Lys Ala Ile Arg Tyr Gln Glu Pro Tyr Ile Tyr

165 170 175

Ala Ser Asn Leu Pro Pro Glu Leu Leu Asn Gln His Asn Glu Leu Ser

180 185 190

Asn Leu Ala Thr Gly Val Glu Ile Arg Ile Thr Pro Phe Leu Glu His

195 200 205

Thr Lys Leu Thr Thr Arg Asn Asn Gly Met Asn Val Gly Ala Phe Gly

210 215 220

Lys His Thr Lys Ser Tyr Ala Asp Met Tyr Glu Arg Val Leu Arg Lys

225 230 235 240

Lys Leu Ser Ala Arg Ile Lys Ile Trp Ala Pro Ser Asp Val Arg Ser

245 250 255

Lys Ser Ile Cys Lys Gly Gln Tyr His Leu Arg Lys Ile Ala Ser Pro

260 265 270

Ile Gln Leu Asp Gly Asp Gln Val His Arg Glu Ala Asp Ser Ala Lys

275 280 285

Trp Ala Leu Val Glu Gly Lys Asn Thr Val Cys Leu Thr Thr Asn Asp

290 295 300

Tyr Lys Thr Thr Glu Lys Arg Ile Pro Gly Ala Ala Val Cys Val Glu

305 310 315 320

Asn Ala Asn Val Tyr Asn Ala Phe Asn Thr Ala Ala Val Asn Val Ala

325 330 335

Ala Cys Asn Met

340

<210> 4

<211> 969

<212> DNA

<213> Artificial Sequence

<400> 4

caaggtgttg caacttgcaa ggcagatgac aatactgatc tcaactggta ttttgtatac 60

aaacctccaa atgctttaca aacaaaaatt atgcagtcgg gacaaaatcc agcttgggca 120

cgttctgcac agtctatcga gagcaacaac ggccattcaa tagttcgaac aatggcacat 180

tttgtagcag agaaccaaaa catcaaagtt cttgcatata gtgacgatcc accaaatttg 240

ccaccaagaa atgaaaaaag caaagccaaa ggagtacttt taattgataa ttcaggagct 300

aatgcagctg cgtggtttgt gcacacagtg cccaaatttt tatcacatct tggtggttat 360

tcttggccac aaacagaaac agcaaaagga cacatatttt tatgtttatc aatcaatgaa 420

gaatctttaa atgctgtagc taaagcaatt cgataccaag aaccatacat atatgcgagt 480

aatttacctc ctgaactttt gaatcaacat aatgaacttt cgaatttggc gacaggagtt 540

gagattcgca taacaccttt tctggagcat acaaaattaa caacaagaaa taatggcatg 600

aatgttggag cttttggaaa acacacaaaa tcatatgcag atatgtatga aagagttctg 660

agaaaaaaac tttctgcaag aatcaaaata tgggcacctt ctgatgtaag atcgaagtca 720

atttgtaagg gacaatacca tcttcgaaaa attgcttctc caatacagct tgatggtgat 780

caagtgcatc gtgaagctga cagtgcaaaa tgggcattag tggaaggaaa gaacacagta 840

tgtcttacaa caaatgatta taagactact gaaaaacgga ttccgggagc tgctgtttgt 900

gttgaaaatg ctaatgttta taacgctttc aatacagcag cagttaatgt tgcagcatgt 960

aatatgtaa 969

Claims

1. The application of the protein for detecting early-stage trichinosis in preparing an immunochromatographic test strip for detecting sheep trichinosis is characterized in that the test strip comprises a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the binding pad, the nitrocellulose membrane and the water absorption pad are sequentially and fixedly connected to the bottom plate, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is close to one end of the binding pad, the quality control line is close to one end of the water absorption pad, and a second protein shown as SEQ ID NO.3 is coated at the detection line;

the binding pad is coated with a microsphere marked mouse IgG antibody and a first protein shown as SEQ ID NO. 1;

the quality control line is coated with goat anti-mouse IgG antibody;

detecting the trichina antibody in the sample according to the specific reaction of the antigen and the antibody and the immunochromatography analysis technology based on a double-antigen sandwich immunochromatography method; comprises coating a recombinant antigen with an amino acid sequence shown as SEQ ID NO.3, namely a second protein serving as a coating antigen, on a detection line of an immunochromatographic test strip; the binding pad of the immunochromatographic test strip is coated with a recombinant antigen with an amino acid sequence shown as SEQ ID NO.1, namely a first protein which is used as a labeled antigen.

2. The use according to claim 1, wherein,

the coating concentration of the antigen protein at the detection line is 2mg/mL.

3. The use according to claim 1, wherein,

in the conjugate pad, the concentration of the mouse IgG label was 1mg/mL, and the concentration of the label of the first protein was 50. Mu.g/mL.

4. The use according to claim 1, wherein,

the coating concentration of the goat anti-mouse IgG is 2mg/mL.