CN115932255A - Fluorescent microsphere immunochromatography detection kit for goat pox virus antibody and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of biotechnology and animal epidemic disease diagnosis, in particular to a goatpox virus antibody fluorescent microsphere immunochromatography detection kit and a preparation method thereof. The kit at least comprises a test strip, wherein the test strip sequentially comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad from a sample adding end; the time-resolved fluorescent microspheres marked with goat pox truncated protein and modified by carboxyl are sprayed on the bonding pad, and the nucleotide sequence of the goat pox truncated protein is shown as SEQ ID NO:1, the amino acid sequence of the goat pox truncated protein is shown as SEQ ID NO:2 is shown in the specification; the nitrocellulose membrane is provided with a detection line and a quality control line, and the detection line close to the sample adding end is coated with goat pox truncated protein; the quality control line close to the end of the absorbent pad is coated with a monoclonal antibody of the goat pox truncated protein. The kit has the advantages of rapidness, sensitivity, accuracy, stability and the like, and is suitable for detecting large-scale clinical samples.

Description

Fluorescent microsphere immunochromatography detection kit for goat pox virus antibody and preparation method thereof

Technical Field

The invention relates to the technical field of biotechnology and animal epidemic disease diagnosis, in particular to a fluorescent microsphere immunochromatography detection kit for a goat pox virus antibody and a preparation method thereof.

Background

The goat pox is an acute, hot and highly contact infectious disease caused by goat pox virus (GTPV), and can cause symptoms such as variola on the skin, the mucous membrane of the digestive tract and the respiratory tract of a diseased goat after infection, and is the most serious pox disease of animals. The disease belongs to a national animal epidemic disease, has great harmfulness, has high infection rate and disease death rate of flocks of sheep, and can reach 100 percent of death rate of infected lambs. The capripoxvirus is distributed worldwide, has high transmission speed and high morbidity and mortality, and brings serious loss to the sheep industry. The use of the goat pox live vaccine effectively controls the epidemic of the goat pox; the bovine sarcoidosis virus and the capripoxvirus belong to the poxviridae and the capripoxvirus, and the capripoxvirus live vaccine plays an important role in the prevention and control process of the bovine sarcoidosis. However, due to uncertainty of immunity, sporadic occurrence of goatpox in some regions is caused.

At present, the diagnosis of goat pox is mainly based on clinical symptoms and autopsy lesions, as well as immunological methods and molecular biological methods. The commonly used virus neutralization test method has the defects of long period, low specificity, low sensitivity and the like; the Chinese patent application CN102313803A discloses a goat pox positive indirect hemagglutination diagnostic reagent and a preparation method and a detection method thereof, wherein the detection takes 50 minutes; an academic paper published in Chinese veterinary medical science and bulletin "establishment and application of truncated expression of goat pox virus P32 gene and indirect ELISA method" discloses an indirect ELISA method for goat pox virus antibody, which is long in detection time and can only be used for detecting goat pox virus antibody in goat serum samples.

The fluorescent nano material has good application prospect by virtue of high sensitivity and good selectivity, and among various fluorescent markers, the fluorescent microsphere is the most promising in ICA. The fluorescent microsphere immunoassay combines the advantages of fluorescence and immunoassay technology, amplifies weak and complex antigen-antibody reaction signals to easily-detected optical signals, effectively eliminates interference of non-specific fluorescence, and can realize high sensitivity, wide linear range, good stability of reagents, simple operation, high detection efficiency and convenient storage of products.

The invention utilizes the fluorescent microspheres as the marking material to develop the fluorescent microsphere rapid detection kit which is suitable for detecting goat pox virus antibodies in field detection, is simple to operate, is rapid and has high sensitivity, realizes the rapid detection of the goat pox virus antibodies, and can be used for assisting the goat pox live vaccine to better prevent and control the goat pox virus diseases and the bovine nodular skin diseases.

Disclosure of Invention

Aiming at the technical problem of long detection time of the existing goat pox antibody, the invention provides a goat pox virus antibody fluorescent microsphere immunochromatography detection kit and a preparation method thereof.

In a first aspect, the invention provides a goat pox virus antibody fluorescent microsphere immunochromatography detection kit, which at least comprises a test strip, wherein the test strip sequentially comprises a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad from a sample adding end; the time-resolved fluorescent microspheres marked with goat pox truncated protein and modified by carboxyl are sprayed on the bonding pad, and the nucleotide sequence of the goat pox truncated protein is shown as SEQ ID NO:1, the amino acid sequence of the goat pox truncated protein is shown as SEQ ID NO:2 is shown in the specification; the nitrocellulose membrane is provided with a detection line (T line) and a quality control line (C line), and the detection line close to the sample adding end is coated with goat pox truncated protein; the quality control line close to the water absorption pad end is coated with a monoclonal antibody of the murine goat pox truncated protein.

Furthermore, the time-resolved fluorescent microspheres are polystyrene-carboxyl fluorescent microspheres with the average particle size of 200 nm.

Further, the final concentration of the goat pox truncated protein coated by the detection line is 0.2mg/mL, and the final concentration of the mouse antigen monoclonal antibody coated by the quality control line is 0.5mg/mL; the amounts of the detection line and the quality control line sprayed with the film liquid are both 0.5 muL/cm.

Further, the kit also comprises a sample diluent, wherein the sample diluent is 0.01M Tris buffer solution with the pH =8.5, and contains 2% of sucrose, 10% of bovine serum albumin and 0.1% of sodium azide.

Preferably, the absorbent pad is absorbent paper.

Further, still include shell base and shell upper cover, the test paper strip is placed in the shell base according to corresponding the direction, and the shell base passes through the buckle with the shell upper cover and is connected.

In a second aspect, the invention provides a preparation method of a goatpox virus antibody fluorescent microsphere immunochromatography detection kit, which at least comprises the following steps:

preparation of S1 conjugate pad

(1) Preparing fluorescent microspheres: coupling the purified goat pox virus truncated protein to carboxyl-modified time-resolved fluorescent microspheres;

(2) And (3) bonding pad treatment: suspending the goat pox truncated protein labeled carboxyl modified time-resolved fluorescent microspheres with a suspension preservation solution, uniformly spraying the suspension preservation solution on a bonding pad, and drying at 37 ℃ for later use;

suspension storage solution is 0.05M boric acid buffer solution with pH =8.0, and contains 1% bovine serum albumin, 0.1% SDS, 0.1% sodium azide;

s2 coating of detection line and quality control line on nitrocellulose membrane

(1) Preparing the goatpox truncated protein into a final concentration of 0.2mg/mL by using a detection line coating buffer solution, wherein the detection line coating buffer solution is a 0.01M Tris buffer solution with the pH =7.0 and contains 1% of bovine serum albumin and 0.1% of sodium azide;

preparing the mouse original monoclonal antibody into a final concentration of 0.5mg/mL by using a quality control line coating buffer solution, wherein the quality control line coating buffer solution is a 0.01M PBS buffer solution with the pH =7.0 and contains 1% of bovine serum albumin and 0.1% of sodium azide;

(2) And marking the detection line and the quality control line on the nitrocellulose membrane in sequence according to the membrane spraying liquid amount of 0.5 mu L/cm.

Further, the purified goat pox virus truncated protein is obtained according to the following method:

(a) The primer sequences were designed as follows:

an upstream primer: 5 'GGGAATTCCATATGGCAGATATCCCATT-3' (SEQ ID NO: 3);

a downstream primer: 5 'CGCCGCTCGAGTTCAATTATAATGTTATA-3' (SEQ ID NO: 4);

(b) The 4-420bp truncated gene is amplified through PCR, the PCR and pET28a (+) plasmid are subjected to double enzyme digestion, the recovered gene fragment is cloned to the pET28a (+) plasmid to obtain a recombinant expression vector, and the sequencing is correct;

(c) Transformation of recombinant plasmidsE. coliBL21 (DE 3) competent cells, induced expression, and then purified the protein of interest using nickel ion affinity chromatography.

Further, the mouse monoclonal antibody is obtained as follows:

(i) Immunization: the purified truncated protein was mixed with complete Freund's adjuvant in a volume ratio of 1:1, immunizing BALB/c mice of 6-8 weeks old, injecting subcutaneously, and immunizing once every 2 weeks;

(ii) Cell fusion: spleen cells and myeloma cells in BALB/c mice were mixed according to the ratio of cell number 10:1, and fusing according to a conventional method;

(iii) Hybridoma screening and cloning: when the cells grow to 1/3 of the bottom of the hole, measuring cell supernatant by adopting an indirect ELISA method, screening positive holes, and subcloning the positive holes by adopting a limiting dilution method until the positive holes are subcloned to 100 percent;

(iv) Preparing and purifying an antibody: ascites is collected by adopting an in vivo ascites induction method of a mouse, then IgG protein in the purified ascites is extracted by an ammonium sulfate two-step precipitation method, and the ascites is further purified by an SPA column method to obtain the traditional Chinese medicine.

And further, assembling the S3 test strip, namely cutting the test strip into 4mm wide, then putting the test strip into a base of a shell, pressing an upper cover of the shell, putting the shell into an aluminum foil bag, adding a drying agent, and sealing and storing the aluminum foil bag.

In a third aspect, the invention also provides a use method of the goatpox virus antibody fluorescent microsphere immunochromatography detection kit, which specifically comprises the steps of diluting a serum sample by 100 times with a sample diluent, dripping 80 mu L of the diluted sample into a sample adding hole, reading the T/C value on a test strip by using a dry type immunofluorescence analyzer after 10-15min, and judging the result.

The detection card adopts a double-antigen sandwich method, a binding pad of the detection card contains goat pox virus truncated protein marked by time-resolved fluorescent microspheres, the goat pox virus truncated protein is sprayed on a nitrocellulose membrane detection line, and a specific monoclonal antibody of mouse goat pox virus truncated protein is sprayed on a quality control line. When the detection is carried out, if the goat pox virus antibody exists in a serum sample, the Fab fragment of the antibody is specifically combined by the goat pox virus truncated protein marked by the time-resolved fluorescent microspheres in the combination pad, the immune complex flows to the detection line region of the nitrocellulose membrane in a lateral chromatography mode, the other Fab fragment of the antibody is combined with the goat pox virus truncated protein on the detection line, and a detection signal is finished. And (3) marking the goat pox virus truncated protein by unreacted fluorescent microspheres to flow to a quality control line, and combining the goat pox virus truncated protein with the mouse anti-goat pox virus truncated protein monoclonal antibody. The invention realizes the detection of the goat pox virus antibody from the serum and has the advantages of convenient operation, sensitive detection, reliability, stability and the like.

Compared with the prior art, the invention has the following characteristics:

(1) The invention uses the double-antigen sandwich principle, takes the specific monoclonal antibody as a quality control line, and combines the time-resolved fluorescence microsphere technology, thereby greatly improving the sensitivity and stability of the detection card compared with the traditional colloidal gold.

(2) The present invention needs no special equipment and professional technical personnel, can use a commercial dry-type immunofluorescence analyzer or a common ultraviolet lamp to interpret the result, and has simple operation.

(3) The kit can quickly, sensitively and accurately detect the GTPV antibody level in the serum of cattle and sheep, fills the blank in the field at home and abroad, is used for replacing the traditional serological methods such as virus neutralization test and the like, and is suitable for realizing the high-throughput large-scale detection of the GTPV antibody with high sensitivity, strong specificity and good stability.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 shows the results of the detection of the expression of the truncated protein of capripoxvirus in example 1.

Fig. 2 is a schematic view of the structure of the test strip in example 3.

In the figure, 1-sample pad, 2-combination pad, 3-detection line, 4-quality control line, 5-nitrocellulose membrane, 6-water absorption pad and 7-PVC bottom plate.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. 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.

The reagents used in the following examples, which are not specifically described, are all commercial reagents used for fluorescent microsphere labeling and detection of line-sprayed antigen, which is expressed and purified goat pox truncated protein. The mouse monoclonal antibody is obtained by transferring a mouse spleen cell and an SP2/0 cell into an abdominal cavity of a mouse after fusion screening of an immune goat pox virus truncated protein BALB/c mouse spleen cell and the SP2/0 cell, extracting ascites and purifying. The time-resolved fluorescent microspheres are polystyrene-carboxyl fluorescent microspheres with the average particle size of 200 nm.

Example 1 expression and purification of goat pox virus truncated protein

1.1 Cloning of truncated Gene

Designing an upstream synthesis primer F:5 'GGGAATTCCATATGGCAGATATCCCATT-3', a downstream primer R:5 'CGCCGCTCGAGTTCAATTATAATGTTATA-3', the extracted virus genome DNA is taken as a template, and the following PCR amplification system is added: PCR Master Mix (2X) 10. Mu.L, upstream and downstream primers 1. Mu.L each, template DNA 2. Mu.L, H ₂ O6. Mu.L, final volume 20. Mu.L. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30s for 32 cycles; extension at 72 ℃ for 10min and termination at 4 ℃.

1.2 Truncated protein expression and purification

Utilizing Nde I and Xho I double enzyme digestion pET28a (+) plasmid, carrying out gel recovery after 1% gel electrophoresis, connecting the cut-off gene fragment and pET28a (+) plasmid after enzyme digestion recovery by using T4 enzyme, transforming DH5 alpha competence, picking single colony shake bacteria, carrying out bacteria liquid PCR identification, extracting plasmid enzyme digestion identification and obtaining the recombinant expression vector. Transformation of recombinant plasmidsE. coliBL21 (DE 3) competent cells were selected using Carna-resistant LB solid medium and single colonies were picked overnight. The bacterial liquid is shaken to OD according to 1 percent of Canna resistant LB culture medium _600nm =0.6, add IPTG to final concentration 0.1 mM,37 ℃, 180r/min induced expression 6h. Centrifuging the culture solution at 8000r/min and 10 ℃, resuspending the thallus with PBS and ultrasonically crushing, centrifuging to retain the supernatant, washing the precipitate with PBS once, centrifuging to remove the supernatant, and dissolving the precipitate with 8M urea. The supernatant and the precipitate were analyzed by 12% SDS-PAGE electrophoresis, and the protein solution was filtered through a 0.45 μm filter, purified by nickel ion affinity chromatography, and the protein concentration was determined.

The nucleotide sequence of the obtained truncated protein (SEQ ID NO: 1) is:

GCAGATATCCCATTATATGTTATACCAATCGTTGGTCGCGAAATTTCAGATGTAGTTCCAGAATTAAAAAGTGGCAATGATATATTTTATAAAAAAGTTGACACAGTAAAAGATTTTAAAAATTCAGATGTAAATTTTTTTTTAAAAGATAAAAAAGATATCAGTTTATCATATAAGTTCCTTATATGGGAAAAGGTAGAAAAATCAGGAGGTGTTGAAAATTTTACAGAATATTTTTCTGGATTATGTAATGCTCTTTGTACAAAAGAGGTAAAAAGTTCTATTGCAAAACACTTTAGTTTATGGAAATCGTATGCCGATGCTGATATAAAAAATTCTGAGAATAAGTTTATTGTTGTTATAGAAGATGATAACACATTAAAAGATTTAATAACAATATATAACATTATAATTGAA。

the amino acid sequence (SEQ ID NO: 2) of the resulting truncated protein is:

ADIPLYVIPIVGREISDVVPELKSGNDIFYKKVDTVKDFKNSDVNFFLKDKKDISLSYKFLIWEKVEKSGGVENFTEYFSGLCNALCTKEVKSSIAKHFSLWKSYADADIKNSENKFIVVIEDDNTLKDLITIYNIIIE。

EXAMPLE 2 preparation of murine monoclonal antibodies

2.1 Immunization

The purified truncated protein and an equivalent amount of complete Freund's adjuvant are used for immunizing BALB/c mice of 6-8 weeks old, and 40 mu g of the BALB/c mice are injected subcutaneously; three boosts were performed at 2 week intervals, each time 40. Mu.g of antigen was mixed with the same amount of incomplete Freund's adjuvant. When the serum titer of the immune mice reaches more than 1: 100000, the immune mice are directly injected into the abdominal cavity 3d before fusion, and the dose is doubled without adding an adjuvant.

2.2 Cell fusion

Preparation before fusion:

preparation of splenocytes: immunized BALB/c mice were removed under aseptic conditions to remove the spleen, and spleen lymphocyte preparation was completed.

Myeloma cell preparation: two weeks before cell fusion feeding SP2/0 (37 ℃,5% CO) ₂ )。

Preparing feeder cells: one day before cell fusion, healthy BALB/c mice were taken, transferred to a clean bench and the feeder cells were prepared aseptically.

Splenocytes in BALB/c mice were mixed with myeloma cells in their peritoneal cavity according to cell number 10:1, centrifuged at 1000rpm for 5min, the supernatant was discarded, 1mL of 50% PEG was slowly added in a water bath at 37 ℃ for 1min, and the mixture was allowed to stand for 1min. Then 2mL of serum-free medium was added slowly over 2min, followed by 8mL of serum-free medium slowly over 2min, to stop the effect of PEG,centrifuging at 1000rpm for 5min, discarding supernatant, adding 10mL of serum, adding sterilized HAT medium, mixing, pouring into cell culture dish (containing feeder cells), and reacting at 37 deg.C and 5% CO ₂ Culturing in an incubator. After 1 week, the single cell pellet dispersed in the plate was aseptically aspirated into a 96-well plate culture medium and cultured.

2.3 Hybridoma screening and cloning

When the cells grow to 1/3 of the bottom of the hole, the indirect ELISA method is used for screening, and hybridoma cells which are detected to be positive are subcloned by a limiting dilution method until the subcloning is positive by 100%.

2.4 Antibody preparation and purification

Injecting Freund's adjuvant into abdominal cavity of mouse for 7 days, inoculating hybridoma cell to induce ascites, collecting ascites when abdomen of mouse is obviously swollen, and centrifuging to remove cells and oil in ascites. Then extracting IgG protein in the purified ascites by using an ammonium sulfate two-step precipitation method, and further purifying the ascites by using an SPA column method to obtain the ascites.

EXAMPLE 3 preparation of the kit

3.1 Preparation of the conjugate pad

And (4) taking 200 muL of time-resolved fluorescent microspheres, and adding 800 muL of 0.05M boric acid buffer solution to obtain a microsphere suspension. And adding 20 mu L of 10mg/mL EDC solution into the microsphere suspension, uniformly mixing, and activating at room temperature for 30min. Centrifuging at 12000r/min at 4 deg.C for 10min, discarding supernatant, redissolving with 1mL 0.05M boric acid buffer solution, and ultrasonically dispersing for 2min; adding 100 μ L of goat pox virus truncated protein (protein concentration 1.2 mg/mL) to the microsphere suspension; shaking the mixture in a shaking table at 220r/min and 25 ℃ for 2h. Adding 100 muL 10% BSA solution, placing in a shaking table at 220r/min and 25 ℃, and sealing for 2h; centrifuging at 12000r/min at 4 deg.C for 15min, discarding supernatant, re-dissolving with 1mL of 0.05M boric acid buffer solution, washing precipitate, re-dissolving with equal volume of 0.1% BSA-containing 0.05M boric acid buffer solution, and ultrasonic dispersing for 2min. Suspending the goat pox truncated protein labeled carboxyl modified time-resolved fluorescent microspheres with a suspension preservative solution (0.05M boric acid buffer solution with pH =8.0, containing 1% bovine serum albumin, 0.1% SDS, 0.1% sodium azide), uniformly spraying onto a binding pad, and drying at 37 ℃ for later use.

3.2 Coating of detection line and quality control line

The goatpox truncated protein was formulated to a final concentration of 0.2mg/mL with assay-line coating buffer, 0.01M Tris buffer pH =7.0, containing 1% bovine serum albumin, 0.1% sodium azide. The murine monoclonal antibody was formulated to a final concentration of 0.5mg/mL using quality control line coating buffer, 0.01M PBS buffer pH =7.0, containing 1% bovine serum albumin, 0.1% sodium azide. And marking the detection line and the quality control line on the nitrocellulose membrane in sequence according to the membrane spraying liquid amount of 0.5 mu L/cm.

3.3 Assembly of reagent strips

Sticking the nitrocellulose membrane 5 on a PVC bottom plate 7, and sticking a water absorption pad 6 at one end close to the quality control line 4 and overlapping the nitrocellulose membrane 5 by 0.5-1mm; pasting the combination pad 2 at one end close to the detection line 3 and overlapping the nitrocellulose membrane 5 for 0.5-1mm; the absorbent pad 6 is stuck on the rear end of the combination pad 2 and is overlapped with the combination pad by 0.5-1mm; the distance between the quality control line 4 and the detection line 3 is 3.5-4mm. The obtained test strip comprises a sample pad 1, a combination pad 2, a nitrocellulose membrane 5 and a water absorption pad 6 in sequence from a sample adding end.

The test paper strip is evenly cut into 4mm wide, placed in a clamping groove of a shell base, tightly pressed on a shell upper cover, placed in an aluminum foil bag, and added with a drying agent for sealed storage.

The use method of the kit comprises the following steps: serum samples were diluted 100-fold with sample diluent (0.01M Tris buffer pH =8.5, containing 2% sucrose, 10% bovine serum albumin, 0.1% sodium azide), 80 μ L was dropped into the wells, and the results were determined with a dry immunofluorescence analyzer or a normal uv lamp after 10-15 min. The dry type immunofluorescence analyzer judges according to the T/C value, the T/C value is larger than 0.1, and the dry type immunofluorescence analyzer judges to be positive, otherwise, the dry type immunofluorescence analyzer judges to be negative; when the result is judged by common ultraviolet, if both the C line and the T line have fluorescent strips, the result is judged to be positive; if the C line has a fluorescence band and the T line has no fluorescence band, judging the result as negative; if the line C has no fluorescent strip, the detection result is invalid.

Example 4: performance determination of goat pox antibody fluorescent microsphere immunochromatographic test strip

4.1 Sensitivity of the composition

Referring to the using method of the kit in the embodiment 3, the kit in the embodiment 3 is used for detecting GTPV positive goat serum, and when the serum is diluted to 1600 times, the detection result is still positive; while the maximum dilution tested in the serum neutralization test was 400.

4.2 Specificity of

Referring to the using method of the kit in the embodiment 3, the kit in the embodiment 3 is respectively used for detecting sheep parainfluenza, peste des petits ruminants, foot and mouth disease, goat pox virus positive serum and goat pox virus positive serum, the results are shown in table 1, only the T/C value of the detection result of the goat pox virus positive serum and the goat pox virus positive serum is larger than 0.1, and the rest is negative, which indicates that the test strip has good specificity.

TABLE 1 results of specificity test

4.3 Stability of

Referring to the kit usage of example 3, three clinical sheep serum samples were tested and the results were read at 10min and 15min, three times per data read, and compared by indirect ELISA. The data read at two time points of 10min and 15min can be observed from the data, the detection data of the fluorescent microsphere immunochromatographic test strip is relatively stable, the variation coefficient is between 0.32% and 1.23%, the detection data of the indirect ELISA method is relatively obvious in change, and the variation coefficient is between 1.5% and 3.04%.

TABLE 2 stability test results of the kit and ELISA test data of the present invention

4.4 Detection application of clinical samples

30 bovine serum samples of the vaccines of the goat pox immunization are detected by using the kit of the embodiment 3 according to the using method of the kit of the embodiment 3, and the positive rate is 100%. Meanwhile, an indirect ELISA method is used for comparison, 5 parts of negative serum (sheep serum of the vaccines of goat pox immunization) detected by 70 parts of positive serum by the ELISA method is detected, and the positive coincidence rate is 92.86 percent; 4 positive samples are detected in 50 negative sera, and the negative coincidence rate is 92%; the overall percent compliance was 92.5%. The kit can be used for antibody detection after immunization of the goat pox vaccine, and can assist in prevention and control of goat pox and bovine nodular skin diseases.

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention.

Claims (8)

1. A kit for detecting goat pox virus antibody fluorescent microsphere immunochromatography is characterized by at least comprising a test strip, wherein the test strip sequentially comprises a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad from a sample adding end; the time-resolved fluorescent microspheres marked with goat pox truncated protein and modified by carboxyl are sprayed on the bonding pad, and the nucleotide sequence of the goat pox truncated protein is shown as SEQ ID NO:1, the amino acid sequence of the goat pox truncated protein is shown as SEQ ID NO:2 is shown in the specification; the nitrocellulose membrane is provided with a detection line and a quality control line, and the detection line close to the sample adding end is coated with goat pox truncated protein; the quality control line close to the end of the absorbent pad is coated with a monoclonal antibody of the goat pox truncated protein.

2. The goatpox virus antibody fluorescent microsphere immunochromatography detection kit according to claim 1, wherein the time-resolved fluorescent microspheres are polystyrene-carboxyl fluorescent microspheres having an average particle diameter of 200 nm.

3. The goatpox virus antibody fluorescent microsphere immunochromatography detection kit according to claim 1, wherein the final concentration of the goatpox truncated protein coated by the detection line is 0.2mg/mL, and the final concentration of the murine original monoclonal antibody coated by the quality control line is 0.5mg/mL; the film spraying liquid amount of the detection line and the quality control line is 0.5 mu L/cm.

4. The goatpox virus antibody fluorescent microsphere immunochromatography detection kit according to claim 1, further comprising a sample diluent, a housing base and a housing upper cover; the sample diluent is 0.01M Tris buffer solution with pH =8.5, and contains 2% of sucrose, 10% of bovine serum albumin and 0.1% of sodium azide; the test paper strip is placed in the shell base according to corresponding direction, and the shell base is connected with the shell upper cover through the buckle.

5. The preparation method of the goatpox virus antibody fluorescent microsphere immunochromatography detection kit is characterized by at least comprising the following steps:

preparation of S1 conjugate pad

(1) Preparing fluorescent microspheres: coupling the purified goat pox virus truncated protein to carboxyl-modified time-resolved fluorescent microspheres;

(2) And (3) bonding pad treatment: suspending the goat pox truncated protein labeled carboxyl modified time-resolved fluorescent microspheres with a suspension preservation solution, uniformly spraying the suspension preservation solution on a bonding pad, and drying at 37 ℃ for later use;

suspension storage solution is 0.05M boric acid buffer solution with pH =8.0, and contains 1% bovine serum albumin, 0.1% SDS, 0.1% sodium azide;

s2 coating of detection line and quality control line on nitrocellulose membrane

(1) Preparing goatpox truncated protein into 0.2mg/mL by using detection line coating buffer solution, wherein the detection line coating buffer solution is 0.01M Tris buffer solution with pH =7.0 and contains 1% of bovine serum albumin and 0.1% of sodium azide;

preparing the mouse original monoclonal antibody into a final concentration of 0.5mg/mL by using a quality control line coating buffer solution, wherein the quality control line coating buffer solution is a 0.01M PBS buffer solution with the pH =7.0 and contains 1% of bovine serum albumin and 0.1% of sodium azide;

(2) And marking the detection line and the quality control line on the nitrocellulose membrane in sequence according to the membrane spraying liquid amount of 0.5 mu L/cm.

6. The method according to claim 5, wherein the purified truncated protein of capripoxvirus is obtained by:

(a) The primer sequences were designed as follows:

an upstream primer: 5 'GGGAATTCCATATGGCAGATATCCCATT-3';

a downstream primer: 5 'CGCCGCTCGAGTTCAATTATAATGTTATA-3';

(b) The 4-420bp truncated gene is amplified through PCR, the PCR and pET28a (+) plasmid are subjected to double enzyme digestion, the recovered gene fragment is cloned to the pET28a (+) plasmid to obtain a recombinant expression vector, and the sequencing is correct;

(c) Transformation of recombinant plasmidsE. coliBL21 (DE 3) competent cells, induced expression, and then purified the protein of interest using nickel ion affinity chromatography.

7. The method of claim 6, wherein the murine monoclonal antibody is obtained by the following method:

(i) Immunization: the purified truncated protein was mixed with complete Freund's adjuvant in a volume ratio of 1:1, immunizing BALB/c mice of 6-8 weeks old, injecting subcutaneously, and immunizing once every 2 weeks;

(ii) Cell fusion: spleen cells and myeloma cells in BALB/c mice were mixed in a cell count of 10:1, and fusing according to a conventional method;

(iii) Hybridoma screening and cloning: when the cells grow to 1/3 of the bottom of the hole, measuring cell supernatant by adopting an indirect ELISA method, screening positive holes, and subcloning the positive holes by adopting a limiting dilution method until the positive holes are subcloned to 100 percent;

(iv) Preparing and purifying an antibody: ascites is collected by adopting an in vivo ascites induction method of a mouse, then IgG protein in the purified ascites is extracted by an ammonium sulfate two-step precipitation method, and the ascites is further purified by an SPA column method to obtain the traditional Chinese medicine.

8. The method of claim 5, further comprising assembling the test strip S3 by cutting the test strip to a width of 4mm, placing the cut test strip in a base of a housing, pressing a top cover of the housing, placing the test strip in an aluminum foil bag, and adding a desiccant for sealing and storing the test strip.

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