CN111518188A - Specific detection antigen of echinococcosis granulosus of cattle and application thereof - Google Patents

Abstract

The invention discloses a specific detection antigen of echinococcosis granulosus of cattle and application thereof, wherein the specific antigen of echinococcosis granulosus of cattle is a protein (a1) or (a2) as follows: (a1) protein with amino acid sequence shown as SEQ ID.1; (a2) and (b) a protein which has the serine protease inhibitor effect and can be specifically combined with the bovine granulomatosis serum antibody by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein defined in (a 1). The invention expresses the Kazal-type serine protease inhibitor domain-conjugation protein recombinant protein and is used as a test strip detection line coating antigen; meanwhile, the time-resolved fluorescence immunochromatography technology is combined with the characteristics of immune labeling and immunochromatography, and the time-resolved fluorescence microspheres are used as tracers to prepare the immunochromatography test strip which is simple to operate clinically, rapid in diagnosis, high in sensitivity and strong in specificity.

Description

Specific detection antigen of echinococcosis granulosus of cattle and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a specific detection antigen of echinococcosis granulosus of cattle and application thereof.

Background

Echinococcosis granulosa is a serious zoonotic disease in humans and animals, and humans or ruminants develop their disease primarily by eating eggs of Echinococcus granulosus by mistake, which is called "worm cancer". At present, research on echinococcosis granulosa diagnosis technology is less, the most common diagnosis method is to examine the polypide by dissection, with the development of molecular technology and immunological technology, there are molecular diagnoses such as LAMP and immunological diagnosis such as ELISA kit method and colloidal gold, but from clinical application, the methods have low detection accuracy and low convenience, and cannot be convenient for basic-level workers to operate and detect. When animals are infected with echinococcus granulosus, the traditional diagnosis method is diagnosis at slaughter, however, during slaughter, the typical saccular tissue may be granuloma caused by inflammation, resulting in wrong diagnosis; serological diagnosis includes colloidal gold immunochromatography and enzyme-linked immunosorbent assay. These methods generally use natural spine balloon fluid, but the use of natural spine balloon fluid has the disadvantages of being susceptible to cross-reactivity with serum from other diseased animals, expensive to prepare, and difficult to commercialize. Although echinococcosis granulosa has been studied in human diagnosis, there are few studies on the diagnosis of animals, and when animals are naturally infected with the disease, the antibody response is not obvious, which is liable to cause diagnosis errors. Therefore, the immune diagnosis of echinococcus granulosus is improved, a reliable antigen is searched, and the establishment of a high-efficiency, accurate, good-specificity and simple diagnosis technology is of great importance.

Disclosure of Invention

Therefore, the invention provides a specific detection antigen for echinococcosis granulosus bovis and application thereof.

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

a protein, such as the protein of (a1) or (a 2):

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

(a2) and (b) a protein which has the serine protease inhibitor effect and can be specifically combined with the bovine granulomatosis serum antibody by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein defined in (a 1).

Nucleic acid molecules encoding the proteins described above are also within the scope of the invention.

The nucleic acid molecule is a gene encoding the protein, and the gene is a DNA molecule described in any one of the following:

(b1) DNA molecule with the coding sequence shown in SEQ ID NO. 2;

(b2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (b1) and which encodes the protein of claim 1;

(b3) a DNA molecule having 90% or more homology with the DNA molecule defined in (b1) or (b2) and encoding the protein of claim 1.

The application of the protein in the preparation of an immunochromatographic test strip for detecting echinococcosis granulosa of cattle, wherein the test strip comprises a bottom plate, and a nitrocellulose membrane and a water absorption pad which are sequentially and fixedly connected with a sample pad, a combination pad, a detection line and a quality control line on the bottom plate, the detection line is close to one end of the combination pad, the quality control line is close to one end of the water absorption pad, and the detection line is coated with the protein of claim 1;

the combination pad is coated with an anti-bovine IgG antibody marked by microspheres;

and a rabbit anti-goat IgG antibody is coated on the quality control line.

The detection antigen is a protein with 6 histidines added at the amino terminal of the amino acid sequence shown in SEQ ID.1.

The coating concentration of the antigen protein at the detection line is 1 mg/mL.

The invention has the following advantages:

the invention expresses the Kazal-type serine protease inhibitor domain-conjugation protein recombinant protein and is used as a test strip detection line coating antigen; meanwhile, the time-resolved fluorescence immunochromatography technology combines the characteristics of immune labeling and immunochromatography, and the prepared immunochromatography test strip is simple in clinical operation, rapid in diagnosis, high in sensitivity and strong in specificity by taking the time-resolved fluorescence microspheres as tracers.

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 should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 is a double restriction enzyme-digestion-verified electrophoresis of the recombinant plasmid of Kazal-type serine protein inhibitor domain-ligation protein-pGEX-4T-1 of the present invention;

FIG. 2 is an SDS-PAGE electrophoresis of the Kazal-type serine protein inhibitor domain-conjugation protein-GST recombinant protein of the present invention;

FIG. 3 is a Western-blot identification chart of the Kazal-type serine protein inhibitor domain-conjugation protein-GST recombinant protein of the present invention;

fig. 4 is a schematic structural diagram of the time-resolved fluorescence immunochromatographic test strip of the present invention, in which: 1-PVC base plate; 2-sample pad; 3-a conjugate pad; 4-absorbent paper; 5-nitrocellulose membrane; 6-detection line T; 7-quality control line C;

fig. 5 is a diagram of a specific detection result of the time-resolved fluorescence immunochromatographic test strip of the present invention, in which: 1-echinococcosis granulosa positive serum of cattle, 2-fascioliasis of cattle liver positive serum, and 3-echinococcosis cerebri positive serum;

fig. 6 is a diagram of a specific detection result of the time-resolved fluorescence immunochromatographic test strip of the present invention, in which: 1-4: diluting serum according to the ratio of 1:500, 1:1000, 1:2000 and 1:4000 in sequence; and 5 is a sample diluent.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of a Kazal-type serine protease inhibitor domain-conjugation protein recombinant protein

1. Construction of pGEX-4T-1-Kazal-type serine protease inhibitor domain-ligation protein expression vector

The Kazal-type serine protease inhibitor domain-containing protein belongs to SPI family, has the function of serine protease inhibitor, and has the functions of regulating inflammatory reaction, innate immunity and antibacterial defense in organisms. The amino acid sequence of the Kazal-type serine protease inhibitor domain-contacting protein is shown as SEQ ID NO.1, and the gene sequence for coding the protein is shown as SEQ ID NO. 2.

Constructing prokaryotic expression vector pGEX-4T-1-Kazal-type serine protease inhibited domain-contacting protein. The expression vector is synthesized by Shanghai Biotechnology Limited, and the expression vector is subjected to double enzyme digestion verification.

The double digestion procedure was as follows:

(1) centrifuging the expression vector freeze-dried powder filled with the target gene at 3000 rpm/normal temperature for 1 min.

(2) With 50. mu.L sterile ddH₂Dissolving the freeze-dried powder by using a vortex instrument, gently mixing the mixture, and instantly separating the mixture for 30 seconds by using a palm centrifuge.

(3) The concentration was measured by NANODROP.

(4) The expression vector plasmid pGEX-4T-1-zal-typeserine protease inhibitor domain-ligation protein was digested by restriction enzymes BamHI and NotI in a double digestion system as shown in Table 1 under 37 ℃/10 min.

TABLE 1

pGEX-4T-1-Kazal-type serine protease inhibitor domain-ligation protein recombinant expression plasmid, the target gene is 756bp, the plasmid is identified after being cut by BamHI/NotI restriction enzyme, and the double-enzyme cutting verification result is shown in figure 1.

Transferring the constructed prokaryotic expression vector pGEX-4T-1-Kazal-type serine protease inhibition-conjugation protein into BL21(DE3) expression competence, performing small-amount induced expression of protein to determine culture conditions, performing large-amount induced expression of protein, eluting glutathione-Sepharose-4B combined with the protein by using 20mM reducing glutathione, and further obtaining the high-purity recombinant antigen.

2. Protein expression purification

1) After double enzyme digestion verification, the recombinant plasmid is transformed to express competence

(1) 50 μ L of ice-cooled BL21(DE3) was placed in a 1.5mL sterile centrifuge tube, thawed in an ice box (without manual thawing), 5 μ L of the expression plasmid was pipetted into the tube and allowed to stand on ice for 30 min.

(2) After ice-bath, the centrifuge tube was placed in a water bath with a float and heat-shocked at 42 ℃ for 45s, and then the tube was transferred to ice and ice-washed for 2 min.

(3) In a sterile ultra clean bench, 500. mu.L of sterile liquid LB medium 400-.

(4) The LB solid plate with corresponding resistance is preheated at 37 ℃, and is taken out for standby after the water vapor in the plate is completely volatilized.

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

(6) The cells were incubated overnight in a 37 ℃ incubator.

2) A small amount induction expression step of GST tag recombinant protein of Kazal-type serine protease inhibitor domain-contacting protein-pGEX-4T-1:

(1) a single colony of transformed BL21(DE3) was picked up with a sterile white pipette tip, and after correct PCR, it was inoculated into 10mL of liquid LB medium containing the corresponding resistance at 200rpm in a 37 ℃ incubator overnight.

(2) The next day, after the bacterial suspension was maintained in the sterile operating table, 100. mu.L of the bacterial suspension was pipetted and inoculated into 10mL of LB medium containing relatively resistant medium, cultured at 37 ℃ and 200rpm, and the OD of the bacterial suspension was determined_600nmWhen the concentration is 0.6-0.8, IPTG induction can be added.

(3) Before adding the protein inducer, 100. mu.L of uninduced whole bacteria were aspirated in a sterile operating table as a negative control. Then, 0.1M IPTG inducer was added to the mixture so that the final concentration of IPTG was 0.1mM, 0.3mM, and 0.6mM, and induction was carried out at different temperatures (16 ℃ C., 22 ℃ C., 32 ℃ C.).

(4) Collecting bacterial liquid which is cultured for 22h at a low temperature of 16 ℃, 18h at a low temperature of 22 ℃ and 8h at a constant temperature of 32 ℃, placing the collected bacterial liquid in a 15mL enzyme removing tube, centrifuging for 15min at 4000rpm of a 4 ℃ centrifuge, discarding supernatant, suspending bacterial precipitates by 1mL phosphate buffer solution, taking 50 μ L of the bacterial liquid as a whole bacterial sample after induction, crushing the rest bacterial liquid by a small ultrasonic crusher, wherein the crushing power of the instrument is 25w, performing ultrasonic treatment for 2s, stopping for 3s, observing the clear bacterial bodies, placing the crushed bacterial liquid in a 1.5mL EP tube, centrifuging for 10min at 5000rpm in a 4 ℃ centrifuge, taking 100 μ L of the supernatant (supernatant after induction), discarding the supernatant, suspending the precipitates by 1mL phosphate buffer solution, and taking 100 μ L of the supernatant after induction.

(5) Adding supernatant, precipitate and whole bacteria at different temperatures and different IPTG concentrations into 20 μ L1 × SDS-PAGE protein loading buffer respectively, boiling in 100 deg.C boiling water for 10min, and taking out.

(6) And (3) respectively taking 10 mu L of prepared whole bacteria, supernatant and precipitated protein samples with different temperatures and different IPTG concentrations, loading the 10% SDS-PAGE protein gel electrophoresis with the setting power of a protein electrophoresis tank as follows: voltage 150V and time 50 min.

(6) The cut protein gel was placed in a protein staining cassette into which G250-Coomassie brilliant blue staining solution was previously poured, and shaken on a horizontal shaker for 20 min.

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

3. Large-scale expression and purification of GST tag recombinant protein of Kazal-type serine protease inhibitor domain-contacting protein-pGEX-4T-1

Cross-streaking glycerol on Amp resistant culture dish, culturing at 37 deg.c in constant temperature incubator overnight, and referring to GST label small amount inducing expression:

(1) pre-cooling the cooler at 3 deg.C 30min in advance, starting the high-pressure crusher, and buffering with 75% alcohol and ddH₂O, PBS passing through the crushing tube in sequence, and exhausting gas and waste liquid after two times.

(2) Adding a proper amount of PBS according to the amount of the thallus precipitate, completely suspending the thallus precipitate by using a vortex device, pouring the thallus precipitate into a high-pressure crushing tube, repeatedly crushing for 3-4 times, centrifuging for 5min by using an ultra-high-speed 4 ℃ centrifuge at 12000rpm, and reserving the supernatant for later use.

(3) And (3) rinsing the empty purification column twice with PBS, reserving a small amount of PBS, sucking a proper amount of Glutathione SepHarose4B according to the amount of supernatant, slowly adding the supernatant into the purification column, opening a bottom switch when the Glutathione SepHarose4B completely sinks to the bottom, discharging waste liquid, passing the column through PBS with 5 times of column volume to wash the filler, and washing for 3 times.

(4) Adding the supernatant collected in the step (2) into the treated Glutathione SepHarose4B filler, placing on a horizontal rotator, and slowly operating at 4 ℃ for 2 h.

(5) And pouring the mixture of the combined supernatant and the glutaminone SepHarose4B into a purification column, opening a bottom switch after the glutaminone SepHarose4B is completely precipitated, adjusting the flow rate, and finishing column passing when the glutaminone SepHarose4B is completely in the purification column.

(6) Unbound heteroproteins were washed off with sterile PBS, 5mL each time, glutaminone SepHarose4B was gently pipetted up and the column was rotated slightly at an angle for 1-2min, after which time the glutaminone SepHarose4B was completely at the bottom of the column, the switch was opened to drain the PBS and washed 6 times.

(7) First, 1mL of GST eluent was added, Glutathione SepHarose4B was gently blown up by a pipette, and the eluent was collected after the column was slightly tilted and rotated for 1-2 min. Next, after 2mL of GST eluent was added, the previous steps were repeated to collect the eluent. And (4) determining whether to perform the third elution or not after the concentration of the last elution is measured by using the Nanodrop.

(8) And (3) carrying out SDS-PAGE protein electrophoresis on the purified target protein, the impurity washing solution and the eluted Glutathione SepHarose 4B.

4. Western-blot identification of GST-tag recombinant protein of Kazal-type serine protease inhibitor domain-contacting protein-pGEX-4T-1

(1) SDS-PAGE run gel

(2) Cutting a PVDF film with proper size according to the size of the protein adhesive, activating with methanol for 2min, cutting qualitative filter paper into proper size, soaking in a film transfer liquid to drive out bubbles between the filter paper and the filter paper, during film transfer, horizontally placing a black surface of a clamp below, sequentially placing a water absorption net, 3 layers of qualitative filter paper, the protein adhesive, the PVDF film, 3 layers of qualitative filter paper and a water absorption net above, closing the clamp after no bubble is generated, placing the clamp into a film transfer groove, enabling the black surface of the clamp to face the black surface of the film transfer groove, enabling a transparent surface of the clamp to face a red surface of the film transfer groove, checking positive and negative electrodes, and starting to transfer the film, wherein the set power is as follows: the current is 200mA, and the time is 85 min.

(3) After membrane transfer, the PVDF membrane was placed in a previously prepared plastic box containing blocking solution (5% skim milk), placed in a 37 ℃ incubator, and sealed for 1h on a shaker.

(4) Blocking with 5% skim milk containing GST-tag monoclonal antibody (1:10000), and incubating overnight at 4 ℃.

(5) PBST membrane washing, each time for 5min, washing 4 times.

(6) AP goat anti-mouse IgG 1 diluted 15,000 in PBST and incubated at 37 ℃ for 50 min.

(7) And (5) repeating the step.

(8) Preparing a proper amount of BCIP/NET color developing reagent, performing dark color development for 2-3min, observing the result and scanning.

(9) The results of the validation of the Kazal-type server protein inhibitor domain-stabilizing protein-pGEX-4T-1 are shown in FIG. 2 and FIG. 3, wherein FIG. 2 is a SDS-PAGE electrophoresis of the Kazal-type server protein inhibitor domain-stabilizing protein-GST recombinant protein, and FIG. 3 is a Western-blot identification of the Kazal-type server protein inhibitor domain-stabilizing protein-GST recombinant protein.

Example 2 preparation of time-resolved fluoroimmunochromatographic test strip for echinococcosis granulosa in cattle

Preparation of time-resolved fluorescence immunochromatographic test strip for echinococcosis granulosus of cattle

As shown in fig. 4, the time-resolved fluorescence immunochromatographic test strip provided in this embodiment includes a PVC base plate, and a sample pad 2, a binding pad 3, a nitrocellulose membrane 5, and a water absorption pad that are located on the PVC base plate 1. One end of the nitrocellulose membrane is laminated with one end of the combination pad, the other end of the nitrocellulose membrane is laminated with one end of the absorbent paper, and one end of the sample pad is laminated with the other end of the combination pad; the goat anti-bovine IgG antibody marked by the fluorescent microspheres is coated on the combination pad, a detection line 6 is arranged on one side, close to the combination pad, of the nitrocellulose film, a quality control line 7 is arranged close to the water absorption pad, a Kazal-type seraprotease inhibitor domain-containing protein recombinant antigen is coated on the detection line 6, and a rabbit anti-goat IgG secondary antibody is coated on the quality control line. Taking 75 mu L of a serum sample to be detected diluted by a sample diluent, dripping the serum sample to be detected into a sample adding hole of a sample pad, irradiating a test strip by using an ultraviolet lamp after 15min to observe a result, and if a detection line and a quality control line are colored after the sample to be detected is dripped, proving that the sample is positive; if only the quality control line is developed after the sample to be detected is dripped, the sample is proved to be negative.

The process for labeling the goat anti-bovine IgG antibody by the time-resolved fluorescent microspheres comprises the following steps:

adding an appropriate amount of EDC into 100 μ L of the time-resolved fluorescent microspheres, placing the mixture in a vortex oscillator, oscillating and mixing uniformly, and incubating for 30min at room temperature. And (3) diluting the goat anti-bovine IgG antibody with the required dosage to 500 mu L by using 0.01M phosphate buffer solution, adding the mixture into the treated time-resolved fluorescent microspheres after uniformly mixing, immediately oscillating, uniformly mixing, placing the mixture in a rotary mixer, and incubating for 1h at room temperature in a dark place. After the reaction is completed, 2% BSA is added, and the mixture is placed in a rotary mixer and incubated for 1h at room temperature in the dark. Centrifuge at 12000rpm for 20 min. Removing supernatant to obtain concentrated goat anti-bovine IgG antibody, adding appropriate amount of time-resolved fluorescent microsphere working solution, and storing at 4 deg.C under sealed condition in dark place.

Secondly, determining the optimal detection reaction conditions of the time-resolved fluorescence immunochromatographic test strip for the echinococcosis granulosa of the cattle:

1. precondition for optimizing time-resolved fluorescence immunochromatographic test strip

The reaction conditions of the test strip of this example were: the sample adding amount of the positive sample and the sample adding amount of the negative sample are both 75 mu L, wherein the ratio of the serum to the diluent is 1:2, and the reaction time is 15 min. The coating parameter is 0.75 mu L/cm, and the coating is sprayed on a nitrocellulose membrane to coat the antigen or the antibody, so as to form a detection line T line and a quality control line C line.

Determining the coating amount of the Kazal-type serine protease inhibitor domain-contacting protein recombinant antigen: spraying 1mg/mL Kazal-type serieprotease inhibitor domain-conjugation protein recombinant antigen on a detection line T line by using a three-dimensional gold spraying and membrane scribing instrument, and respectively preparing into two groups, wherein each group is three chromatographic test strips, namely a positive sample group. The detection line C is respectively sprayed with rabbit anti-goat IgG secondary antibody of 1mg/mL, 2mg/mL and 3 mg/mL. Coating the nitrocellulose membrane by the same coating parameters to form a detection line T line and a quality control line C line, and drying in an oven with the temperature of 45 +/-1 ℃ and the humidity of less than or equal to 35% for 16 h. And preparing the time-resolved fluorescence immunochromatographic test strip for the echinococcosis granulosus of the cattle. And reading the detection values of the T line and the C line on a fluorescence immunoassay analyzer, and calculating T/C to determine the optimal antigen-antibody coating amount. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

As shown in Table 1, the results of the detection of the amount of the recombinant antigen coating of the present invention, wherein N represents a negative serum sample; p represents a positive serum sample. It was found that the optimal coating amount was obtained when the concentration of rabbit anti-goat IgG was 2.0 mg/mL.

TABLE 1

2. Determination of the concentration of the labeled antigen

Using the same labeling method, labeling different amounts of goat anti-bovine IgG by using the processed time-resolved fluorescent microspheres respectively, wherein the labeling amounts are respectively as follows: 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL. Diluting the echinococcosis granulosa with 4 times of dilution concentration to prepare a time-resolved fluorescence immunochromatographic test strip for the echinococcosis granulosa, reading detection values of a detection line T line and a quality control line C line on a fluorescence immunoassay analyzer, and calculating 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 the negative sample was added dropwise, the T/C value was minimal.

As shown in table 2, the detection results of the concentration of the labeled recombinant antigen, wherein N represents a negative serum sample; p represents a positive serum sample. The labeled antigen goat anti-bovine IgG concentration was found to be the optimal conjugate pad labeling concentration when it was 50. mu.g/mL.

TABLE 2

3. Determination of dilution ratio of fluorescent labeling solution

Diluting the marked fluorescent microspheres by 2 times, 4 times and 8 times with 3 dilutions respectively. And preparing the time-resolved fluoroimmunochromatographic test strip for the echinococcosis granulosa of the cattle, reading detection values of a detection line T line and a quality control line C line in a fluoroimmunoassay analyzer, and calculating T/C to determine the optimal dilution ratio of the fluorescent labeling solution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

As shown in table 3, the results of the measurement of the dilution ratio of the fluorescent labeling solution, wherein N represents a negative serum sample; p represents a positive serum sample. It was found that the optimum dilution ratio of the fluorescent labeling solution was the optimum dilution ratio when the dilution ratio of the fluorescent labeling solution was 4 times.

TABLE 3

4. Determination of fluorescent working fluid

Selecting proper fluorescent microsphere suspension according to the determined dilution ratio of the fluorescent labeling solution, wherein the proper fluorescent microsphere suspension is divided into three groups: the first group is: 0.05m tris-HCl, 0.9% NaCl, 0.05% BSA, 0.05% Tween20, adjusted to pH 7.9; the second group is: 0.01M phosphate buffer; the third group is: 0.1M Tris-HCl, 0.1% BSA (5mL), 10% S9, 10% Twen20, 10% PEG12000, 3g trehalose. The time-resolved fluoroimmunoassay test strip for the echinococcosis granulosus of the cattle is prepared, the detection values of a T line and a C line are read by a fluoroimmunoassay analyzer, and the T/C is calculated to determine the optimal fluorescent working solution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

As shown in table 4, the fluorescence working solution assay results, wherein 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

5. Determination of sample pad treatment fluid

In order to ensure that the sample can be better combined with the antibody marked by the fluorescent microspheres in the combined pad, three sample pad treatment solutions are prepared and divided into three groups. The first group is: 1% BSA, 0.1% Triton-100, 0.02mol/L phosphate buffer pH 7.4; the second group is: 0.1M Na₂B₄O₇·10H₂O、1％PVP、0.2％Casein-Na、1％Triton-X100、1％Tetronic 1307、0.2％NaN₃Adjusting the pH value to 9.3; the third group is: 0.5M boric acid buffer, 1% triton x-100, 1% PVP, 2% NaCl, adjusted to pH 9.0. And preparing the time-resolved fluoroimmunoassay test strip for the echinococcosis granulosus, reading the detection values of the T line and the C line in a fluoroimmunoassay analyzer, and calculating T/C to determine the optimal sample pad treatment solution. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

As shown in table 5, the results of the sample pad treatment assay, where N represents a negative serum sample; p represents a positive serum sample. It was found that the second set of sample pad treatment solutions was the best sample pad treatment solution.

TABLE 5

6. Determination of reaction time

Sucking 75 mu L of sample by a pipette, adding the sample into 150 mu L of sample buffer solution, fully and uniformly mixing for 30-60 s, sucking 75 mu L of mixed sample by the pipette, dripping the mixed sample into the sample adding hole of the detection card, and respectively reading after reacting for 10min, 15min and 20 min. And reading the detection values of the T line and the C line in a fluorescence immunoassay analyzer, calculating T/C, and determining the optimal reaction time. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

As shown in table 6, the results of the measurement of the reaction time, wherein N represents a negative serum sample; p represents a positive serum sample. The optimal reaction time of the chromatographic test strip is found to be 15 min.

TABLE 6

7. Determination of sample buffer

To ensure that the antigen and antibody are fully reacted, three sample dilutions were searched, divided into three groups, each: a first group: 0.02M Tris-HCl pH 7.8, 0.9% NaCl, 0.1% BSA, 0.5% Tween-20, 0.1% NaN₃(ii) a Second group: 0.05M Tris-HCl pH 7.8, 0.9% NaCl, 1.5% BSA, 0.01% Tween-20, 0.1% NaN₃(ii) a Third group: PBS buffer pH 7.8. And reading the detection values of the T line and the C line in a fluorescence immunoassay analyzer, and calculating T/C to determine the optimal sample diluent. The conditions selected were: when a positive sample is dripped, the T/C value is maximum; when the negative sample was added dropwise, the T/C value was minimal.

As shown in table 7, the results of the assay in the sample buffer, wherein N represents a negative serum sample; p represents a positive serum sample. The second set of sample buffers was found to be the optimal sample diluent.

TABLE 7

8. Determination of dilution ratio of sample

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

TABLE 8

As shown in table 9, the results of the assay of the sample dilutions, wherein N represents a negative serum sample; p represents a positive serum sample. The optimum dilution ratio was found to be 1:2 (sample volume: sample buffer)

TABLE 9

Test example, the specificity, sensitivity and accuracy of the time-resolved fluorescence immunochromatographic test strip of the present invention were measured

1. The specificity of the time-resolved fluorescence microtitre chromatography test strip

The positive serum of the bovine fasciolosis and the positive serum sample of the bovine echinococcosis are detected, except the positive serum of the bovine echinococcosis granulosa, under a fluorescence immunoassay analyzer, the serum of other non-bovine echinococcosis granulosa pathogens has no signal detected on a T line, and only one band appears on a C line, namely the negative result, wherein the results are shown in figure 5, wherein the positive serum of the 1-bovine echinococcosis granulosa, the positive serum of the 2-bovine fasciolosis and the positive serum of the 3-bovine echinococcosis are shown in the figure.

2. The sensitivity of the time-resolved fluorescence chromatography test strip

Diluting the positive seroline at a ratio of 1:500, 1:1000, 1:2000 and 1:4000, respectively dripping the diluted sample into a sample pad, and observing the fluorescence intensity of the T line under fluorescence immunochromatography after 15 min. The results show that line T, C is clearly visible when the positive serum dilution ratio is 1:2000, with the results shown in fig. 6, where: 1-4: diluting serum according to the ratio of 1:500, 1:1000, 1:2000 and 1:4000 in sequence; and 5 is a sample diluent.

3. The accuracy of the time-resolved fluorescence immunochromatographic test strip

In order to further verify the detection effectiveness of the time-resolved fluoroimmunoassay test strip, 50 parts of positive serum of echinococcosis granulosus of cattle and 50 parts of healthy bovine serum are respectively detected by using two commercial ELISA kits and the method, the results are shown in Table 10, and the detection accuracy based on the time-resolved fluoroimmunoassay test strip is higher as compared with the results of the two commercial ELISA kits.

Watch 10

The time-resolved fluorescence immunochromatographic test strip is strong in specificity, and good in sensitivity and accuracy.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Shenyang agriculture university

<120> specific detection antigen of echinococcosis granulosus of cattle and application thereof

<130>GG20753254A

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>275

<212>PRT

<213>Artificial Sequence

<400>1

Met Leu Ala Ala Lys Ser Met Val Gly Leu Phe Leu Leu Val Leu Phe

1 5 10 15

Ala Leu Ser Ser Arg Glu Ser Asn Ala Glu Glu Ala Gly Glu Thr Pro

20 25 30

Leu Asp Val Cys Ala Ser Cys Asp Glu Ser Lys Cys Pro Pro Val Thr

35 40 45

Met Cys Pro Val Gly Glu Val Lys Asp Tyr Cys Gly Cys Cys Ser Val

50 55 60

Cys Gly Leu Glu Gln Gly His Arg Cys Asn Thr Arg Gln Glu Leu His

65 70 75 80

Asp Met Leu Ser Gly Arg Arg Arg His Gly Tyr Tyr Gly Ala Cys Gly

85 90 95

Lys Asn Leu Glu Cys Gln Pro Arg Thr Asp Val Asp Glu Gln Ser Leu

100 105 110

Gly Glu Glu Asn Ile Cys Val Cys Thr Lys Pro Gly Arg Phe Cys Ala

115 120 125

Ser Asn Gly Glu Thr Tyr Ser Ala Cys Glu Leu Glu Ala Val Gln Ala

130 135 140

Lys Ser Phe Gly Glu Val Phe Leu Ile Ser Tyr Asp Asp Cys Lys Ser

145 150 155 160

Glu Pro Lys Ile Val Ala Ala Ser Glu Ser Gln Arg Val Pro Glu Gly

165 170 175

Asn Arg Thr Thr Phe Trp Cys Glu Ile Lys Gly Tyr Pro Leu Pro Thr

180 185 190

Val Thr Trp Tyr Tyr Phe Ala Pro Gly Gly Ser Tyr Glu Ala Ile Leu

195 200 205

Leu Pro Gly Asp Ser Asp Glu Met Ser Val Ser Leu Arg Gly Ala Pro

210 215 220

Pro Gly Arg Arg Ile Ile Ser His Leu Gln Ile Arg Arg Phe Asp Ile

225 230 235 240

Lys Tyr Glu Gly Ile Tyr Gln Cys Tyr Val Glu Asn Asp Leu Gly Ser

245 250 255

Asp Arg His Asn Ile Thr Ala Ile Tyr Ala Pro Pro Glu Pro Arg Pro

260 265 270

Arg Asp Leu

275

<210>2

<211>828

<212>DNA

<213>Artificial Sequence

<400>2

atgctagccg ccaagtcgat ggttggcctc ttcctcctcg tcctcttcgc tctgagtagt 60

cgtgagtcta acgcagagga ggctggtgaa acaccgctgg atgtctgcgc gtcctgcgat 120

gagtcaaagt gtccccccgt gaccatgtgt cccgtgggag aggtaaaaga ctattgtggc 180

tgttgctcgg tctgcggctt agagcaggga catcgatgca atacgagaca ggaattacat 240

gacatgctct cgggcagacg acgtcacggc tactacggcg cctgcggcaa gaaccttgaa 300

tgtcagcctc gcactgacgt tgatgagcag tcactgggtg aggagaacat ttgcgtgtgc 360

accaagcccg gtcgtttctg cgccagcaac ggagaaacct actcggcttg tgagttggag 420

gccgtgcagg ccaagtcgtt tggtgaggta ttcctcatct cctatgacga ttgtaaatca 480

gagccgaaga ttgtagccgc atcggagtcg cagcgagttc ccgaaggaaa caggacaacc 540

ttctggtgcg aaatcaaggg ttatcccctt ccaacagtca cctggtatta ctttgctccc 600

ggtggatcat atgaggccat tctactcccg ggggactctg atgaaatgag tgtgagtctg 660

cgtggtgcgc caccgggacg tcgcataatc tcccacctgc agatccggag attcgacatc 720

aaatacgagg gcatctacca gtgctacgta gagaacgatc tcggaagtga ccgccacaat 780

atcaccgcca tctacgctcc accggagcct cggcctcgag atctctag 828

Claims (6)

1. A protein, such as the protein of (a1) or (a 2):

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

(a2) and (b) a protein which has the serine protease inhibitor effect and can be specifically combined with the bovine granulomatosis serum antibody by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein defined in (a 1).

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein said nucleic acid molecule is a gene encoding the protein of claim 1, said gene being a DNA molecule of any one of the following:

(b1) DNA molecule with the coding sequence shown in SEQ ID NO. 2;

(b2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (b1) and which encodes the protein of claim 1;

(b3) a DNA molecule having 90% or more homology with the DNA molecule defined in (b1) or (b2) and encoding the protein of claim 1.

4. The use of the protein of claim 1 in the preparation of an immunochromatographic test strip for detecting echinococcosis granulosa in cattle, wherein the test strip comprises a bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane having a detection line and a quality control line, and a water absorbent pad fixedly connected to the bottom plate in sequence, the detection line is near one end of the conjugate pad, the quality control line is near one end of the water absorbent pad, and the detection line is coated with the protein of claim 1;

the combination pad is coated with an anti-bovine IgG antibody marked by microspheres;

and a rabbit anti-goat IgG antibody is coated on the quality control line.

5. The use according to claim 4,

the detection antigen is a protein with 6 histidines added at the amino terminal of the amino acid sequence shown in SEQ ID.1.

6. The use according to claim 4,

the coating concentration of the antigen protein at the detection line is 1 mg/mL.

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