A kit for detecting antibodies to equine infectious anemia virus

Abstract

The application discloses a kit for detecting equine infectious anemia virus antibody and belongs to the field of animal virus antibody detection. The kit comprises equine infectious anemia virus p26-gp90 recombinant protein, mouse IgG and goat anti-mouse IgG, and the equine infectious anemia virus p26-gp90 recombinant protein is composed of an amino acid sequence shown in SEQ ID NO. 1. The kit is used for detecting equine infectious anemia virus antibody, is convenient and fast, has high sensitivity, has no cross reaction with other pathogens, has high specificity, has great clinical significance and wide application prospect.

Description

[0001] Related patents

[0002] This application is a divisional application of Chinese invention patent application No. 202011474580X, filed on December 14, 2020, entitled "A recombinant protein of equine infectious anemia virus p26-gp90 and its preparation method and application". Technical Field

[0003] This invention belongs to the field of animal virus antibody detection, specifically, it relates to a kit for detecting antibodies against equine infectious anemia virus. Background Technology

[0004] Equine Infectious Anemia (EIA) is a contagious disease of horses, mules, and donkeys caused by the equine infectious anemia virus (EIA), a member of the Lentivirinae subfamily of the Retroviridae family. EIA virus belongs to the Lentiviral genus of the Retroviridae family and is the pathogen that seriously harms equines. The main characteristics of EIA are intermittent fever, weight loss, progressive weakness, anemia, hemorrhage, and edema; symptoms gradually lessen or temporarily disappear during periods without fever.

[0005] The agar gel immunodiffusion assay was developed by American veterinary microbiologists L. Coggins and Knox, and is currently listed as one of the specific diagnostic methods for equine infectious anemia (EIA) in my country. In addition, fluorescent antibody staining techniques and neutralization tests, as well as enzyme-linked immunosorbent assays (ELISA), can also be used as auxiliary diagnostic tests for EIA. Furthermore, biological experiments (inoculation of healthy foals) can also be performed. However, the aforementioned immunofluorescence staining techniques and ELISA methods require specific instruments, equipment, and corresponding experimental conditions and skills, making them difficult to promote at the grassroots level. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a recombinant protein of equine infectious anemia virus p26-gp90, comprising the amino acid sequence shown in SEQ ID NO.1.

[0008] In this invention, recombinant protein, also called fusion protein or recombinant fusion protein, is the expression product of two genes recombined by DNA recombination technology.

[0009] The capsid protein (CA) p26 is the main immunogenic protein of equine infectious anemia virus (EIV), which can induce and initiate the body's immune system to produce an immune response. Currently, this protein is widely used as a detection antigen both domestically and internationally. However, if the type of detection antigen used is too singular, there is an inevitable risk of false negatives. To reduce the risk of false negatives, this invention fuses the p26 protein with the full-length protein sequence of another protein, gp90, which can significantly improve the sensitivity of diagnosis. Therefore, fusing the major antigenic epitope of p26 and the full-length sequence of gp90 protein together not only improves the sensitivity of diagnosis but also reduces cross-reactivity with other pathogens.

[0010] In some embodiments of the present invention, preferably, the recombinant protein consists of the amino acid sequence shown in SEQ ID NO.1.

[0011] A second aspect of the present invention provides a gene encoding the recombinant protein described in the first aspect of the present invention, comprising the nucleotide sequence shown in SEQ ID NO.2.

[0012] This gene sequence was designed for expression of the recombinant protein in *E. coli*, and the codons were optimized based on *E. coli*'s codon preferences. Different species use synonymous codons at different frequencies, and this codon preference affects the translation process. If an mRNA contains many clusters of rare codons, it can negatively impact ribosome motility, significantly reducing protein expression levels. Therefore, the gene sequence was codon-optimized for *E. coli* expression, improving protein expression efficiency.

[0013] A third aspect of the present invention provides an expression vector comprising the gene described in the second aspect of the present invention.

[0014] In some embodiments of the present invention, the expression vector is pET30a, which is kanamycin resistant, and the expressed fusion protein has a histidine (His) tag.

[0015] A fourth aspect of the present invention provides a host cell containing the expression vector described in the third aspect of the present invention.

[0016] Furthermore, the host cell is a eukaryotic host cell or a prokaryotic host cell.

[0017] In some embodiments of the present invention, the host cell is a prokaryotic host cell. Preferably, the host cell is *Escherichia coli*, and more preferably, *Escherichia coli* BL21. Expression using *E. coli* has advantages such as short cycle time, low cost, and high expression level.

[0018] A fifth aspect of the present invention provides a method for preparing the recombinant protein described in the first aspect of the present invention, comprising the step of inducing the host cells described in the fourth aspect of the present invention to express the protein.

[0019] Furthermore, the host cell is a eukaryotic host cell or a prokaryotic host cell.

[0020] In some embodiments of the present invention, the host cell is a prokaryotic host cell. Preferably, the host cell is *Escherichia coli*, and more preferably, *Escherichia coli* BL21. Expression using *E. coli* has advantages such as short cycle time, low cost, and high expression level.

[0021] In some specific embodiments of the present invention, the step of inducing Escherichia coli to express proteins is as follows:

[0022] S1, the *E. coli* was cultured in LB medium containing 50 μg / mL kanamycin at 37°C.

[0023] S2. When the OD600 of the E. coli culture medium reaches 0.5-0.7, expression is induced by IPTG at a final concentration of 1 mM. The induction conditions are: 25℃, 200 rpm, 4 h. Using these induction conditions, the recombinant protein can be expressed more slowly, allowing sufficient time for spatial conformation formation, which is very important for the recombinant protein to function.

[0024] S3, centrifuge the culture medium at 7000 rpm for 10 min at 4℃ and collect the bacterial cells;

[0025] S4, use Binding Buffer to lyse the bacterial cells;

[0026] S5, ultrasonic disruption of bacterial cells, conditions: 550w, 2s of sonication, 5s interval, 80-120 times in total;

[0027] S6, centrifuge at 4℃, 12000rpm for 30min and collect the supernatant. The recombinant protein is in the supernatant.

[0028] Preferably, in step S2, induction is performed when the OD of the Escherichia coli culture medium is between 600 and 0.6.

[0029] Preferably, in step S5, the ultrasonic disruption is performed 100 times. Using the disruption method of this invention avoids excessively vigorous disruption that could lead to loss of recombinant proteins.

[0030] In some embodiments of the present invention, a further step of purifying the recombinant protein is included. Various methods can be used to purify the recombinant protein, such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography. In some embodiments of the present invention, affinity chromatography is chosen because the recombinant protein contains a His tag, and one-step purification can achieve high purity.

[0031] In some specific embodiments of the present invention, the supernatant containing the recombinant protein is passed through a Ni column and then eluted with elution buffer to obtain the target protein.

[0032] Preferably, the elution buffer is formulated as follows: 50 mM Tris, 0.2 M NaCl, 0.5 M Midazole, pH 8.0.

[0033] A sixth aspect of the present invention provides the use of the recombinant protein described in the first aspect of the present invention in the preparation of a kit for detecting antibodies against equine infectious anemia virus.

[0034] A seventh aspect of the present invention provides a kit for detecting antibodies against equine infectious anemia virus, comprising the recombinant protein described in the first aspect of the present invention.

[0035] Furthermore, the kit also includes mouse IgG and goat anti-mouse IgG.

[0036] In some embodiments of the present invention, a double-antigen sandwich gold labeling method is used to detect equine infectious anemia virus antibodies.

[0037] In some specific embodiments of the present invention, the kit includes a double-antigen sandwich gold labeling test strip, and the reagent method for the test strip is as follows:

[0038] S1, prepare recombinant protein colloidal gold complex and mouse IgG colloidal gold complex respectively;

[0039] S2, the recombinant protein colloidal gold complex and mouse IgG colloidal gold complex are mixed to prepare a gold-labeled pad;

[0040] S3, using recombinant protein as the detection line and goat anti-mouse IgG as the quality control line, is drawn on a nitrocellulose membrane;

[0041] S4. Filter paper, a polyester plate containing a nitrocellulose membrane, a gold label pad, and a sample pad are mounted on a base plate. A portion of the filter paper is stacked on the polyester plate, a portion of the polyester plate is stacked on the gold label pad, and a portion of the gold label pad is stacked on the sample pad. The polyester plate has a test area and a quality control area. The test area has a detection line (T line), and the quality control area has a quality control line (C line). The detection line is close to the gold label pad, and the quality control line is close to the filter paper, thus preparing the test strip.

[0042] When using this method, add the equine biological sample to the sample pad, let it stand at room temperature for 10 minutes, and then determine the test results. The determination criteria are as follows:

[0043] ① The presence of two bands, one in the control area and the other in the test area, indicates a positive result;

[0044] ② If only one band appears on the control line and no band appears in the test area, the result is negative;

[0045] ③ If no band appears on the control line, it indicates that the test strip is damaged. Regardless of whether a band appears on the test line, a new test strip should be used and the test repeated.

[0046] In some embodiments of the present invention, a positive result for equine infectious anemia virus antibody detection indicates that the equine biological sample contains equine infectious anemia virus antibodies, meaning that the equine has been infected with or has been infected with equine infectious anemia virus.

[0047] In some embodiments of the invention, the biological sample is serum or plasma, or any other bodily fluid that may contain antibodies.

[0048] In this invention, the equine animals are selected from horses, mules, and donkeys.

[0049] Beneficial effects of the present invention

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] p26 and gp90 proteins are the main immunogenic antigens of equine infectious anemia virus and are highly conserved. They can induce and activate the body's immune system to produce an immune response and induce host cells to produce neutralizing antibodies. Therefore, fusing the main antigenic epitope of p26 protein and the full-length sequence of gp90 protein together for expression can not only improve the sensitivity of diagnosis, but also reduce cross-reaction with other pathogens. It has high specificity and has great clinical significance and broad application prospects.

[0052] The colloidal gold-labeled immunoassay method used in this invention is a novel analytical technique that is fast, simple, low-cost, pollution-free, and requires no training. Compared with traditional methods, it is more suitable for on-site testing, with advantages such as short color development time and no need for expensive instruments, and has broad market prospects and application value. Attached Figure Description

[0053] Figure 1 The gel electrophoresis results of the purified equine infectious anemia virus p26-gp90 fusion protein are shown. 1: Loading after cell lysis; 2: Flow bleed; 3: Elution with 50 mM Imidazole; 4: Elution with 0.5 M Imidazole.

[0054] Figure 2 The diagram shows a reagent image of a test strip according to an embodiment of the present invention. 1: Sample pad; 2: Gold label pad; 3: NC membrane; 31: Detection line (T line); 32: Control line (C line); 4: Filter paper; 5: Backing plate.

[0055] Figure 3 This diagram illustrates the results of a test using a test strip according to an embodiment of the present invention. T: test line, C: control line.

[0056] Figure 4 The overall results of testing clinical equine serum samples using the test strips of the present invention are shown. Detailed Implementation

[0057] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0058] Example

[0059] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0061] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0063] Example 1: Construction of the expression vector for the equine infectious anemia virus p26-gp90 fusion protein gene

[0064] The equine infectious anemia virus p26 gene was designed based on the protein sequence of NCBI Gene Bank: ABE03841. The envelope polyprotein gp90 gene was designed based on the protein sequence of NCBI Gene Bank: AAC24024.

[0065] The amino acid sequence of the p26-gp90 recombinant protein fusion protein is as follows (SEQ ID NO.1):

[0066] EFIDGAGNRNFRPLTPRGYTTWVNTIQQHNLLNEASVNLFGILSVDCTSEEMNAFLDVVPGQAGQKQVLLDALDKIAEDWDNRHPLPNAPLVAPPQGPIPMTARFIRGLGVPRERQMEPAFDQFRQTYRQWIIEAMTEGIKVMTGKPKAQNIR QGPKEPYPEFVDRLLSQIESEGHSTEITRFLTDTLTIQNANEECRNAMRHLRPEDSLEEKMYACRDFGSTKLSKNSMAESKEARDQEMNLKEEKEEKRRNDWWKIGMFLLCLAGTTGGILWWYEGLPQQHYIGLVAIGGRLNGSGQSNAIECW GSFPGCRPFQNYFSYETNRSMHMNNNTATLLEAYHREITFIYKSSCTDSDHCQEYQCKKVDLINSSSNSVRVVENETTTEYWGFKWLECNQTENLKTILVPENEMVNINDSDTWIPKGCNETWARVKRCPIDILYGIHPIRLCVQPPFFLVQEK GIANNSRISNCGPTIFLGVLEDNKGVIRGNSTICKVNITEIKRKDYTGIYQVPIFYTCNFTNITSCNNESIISVIMYDTNQVQYLLCNNNNSNNYNCVVQSFGVIGQAHLELPRLNKRIRNQSFNQYNCSINNKTELETWKLVKTSGITPLPIS

[0067] Different species use synonymous codons at different frequencies, and this codon preference affects the translation process. If an mRNA contains many clusters of rare codons, it can negatively impact the speed of ribosome movement, significantly reducing protein expression levels.

[0068] This invention utilizes *Escherichia coli* as an expression system. To obtain higher expression efficiency and higher expression levels, codon optimization was performed during the expression of exogenous proteins. The resulting nucleotide sequence is shown below (SEQ ID NO.2):

[0069]

[0070] The recombinant gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated with the pET30a plasmid to form a recombinant expression vector.

[0071] Example 2: Expression of equine infectious anemia virus p26-gp90 fusion protein

[0072] The equine infectious anemia virus p26-gp90 fusion gene plasmid was transformed into *E. coli* BL21 and plated on LB agar plates containing 50 μg / mL kanamycin (Shanghai Sangon Biotech, catalog number: K0408). The plates were incubated overnight at 37°C. Single colonies were picked and cultured in 300 mL LB medium containing the same concentration of kanamycin at 37°C until the OD600 reached approximately 0.6. Expression was then induced with 1 mM IPTG (Shanghai Sangon Biotech, catalog number: IB0168) at 25°C for 4 h. After induction, the culture was centrifuged at 7000 rpm for 10 min at 4°C, and the bacterial cells were collected.

[0073] Example 3: Purification and refolding of equine infectious anemia virus p26-gp90 fusion protein

[0074] The bacterial cells were lysed using 50 mL of Binding Buffer (50 mM Tris, 0.2 M NaCl, pH 8.0); then sonicated at 550 W for 2 seconds, followed by 5-second intervals, for a total of 100 cycles; finally, the cells were centrifuged at 12000 rpm for 30 minutes at 4°C, and the supernatant was collected. The target protein was in the supernatant. The cells were then purified in one step using a Ni column, eluting the target protein with Elution Buffer (50 mM Tris, 0.2 M NaCl, 0.5 M Imidazole, pH 8.0). The target protein was detected by PAGE gel electrophoresis, and the results are shown below. Figure 1 As shown.

[0075] Depend on Figure 1 It was found that the purified fusion protein had high purity. The purified recombinant protein was dialyzed with dialysis buffer (50 mM Tris, 0.2 M NaCl, pH 8.0), with the dialysis buffer changed every 12 hours for a total of 3 times. The dialyzed protein solution was then filtered through a 0.22 μm filter, and the concentration was determined by the BCA method. It was then stored at -20℃ for later use.

[0076] Example 4: Detection of Equine Infectious Anemia Virus Antibodies by Double Antigen Sandwich Gold Labeling

[0077] 1. Preparation of double-antigen sandwich gold labeling test strip

[0078] 1.1 Firing of Colloidal Gold

[0079] Add 1000 mL of ultrapure water to an Erlenmeyer flask and heat to boiling on a magnetic stirrer. Then add 4 mL of 10% chloroauric acid (Sigma) and 6 mL of 10% trisodium citrate solution. Continue heating to boiling for 5 min. After cooling to room temperature, filter the colloidal gold through a 0.22 μm filter and store at 4 °C for later use.

[0080] 1.2 Labeling of recombinant equine infectious anemia virus p26-gp90 fusion protein

[0081] Take 100 mL of colloidal gold solution and put it into a beaker. Add 0.2 M K2CO3 to adjust the pH of the gold solution to 7.5. After stirring, add 1.5 mg of purified recombinant equine infectious anemia virus p26-gp90 fusion protein. Stir at room temperature for 15 min. Add 1 mL of 10% BSA solution. Stir at room temperature for 15 min. Centrifuge at 12000 rpm for 10 min. Carefully aspirate and discard the supernatant. Dilute the precipitate to 1 mL with gold labeling dilution buffer (20 mM Tris, 1% BSA, 0.03% Proclin 300, pH 8.0). This is the labeled recombinant equine infectious anemia virus p26-gp90 fusion protein colloidal gold complex.

[0082] 1.3 Mouse IgG Marker

[0083] Take 100 mL of colloidal gold solution and put it into a beaker. Add 0.2 M K2CO3 to adjust the pH of the gold solution to 7.0. After stirring, add 1 mg of mouse IgG (Hangzhou LONGi Biotechnology Co., Ltd., catalog number: AS00901). Stir at room temperature for 15 min. Add 1 mL of 10% BSA solution. Stir at room temperature for 15 min. Centrifuge at 12000 rpm for 10 min. Carefully aspirate and discard the supernatant. Dilute the precipitate to 1 mL with gold labeling dilution buffer (20 mM Tris, 1% BSA, 0.03% Proclin 300, pH 8.0). This is the labeled mouse IgG colloidal gold complex.

[0084] The gold-labeled complex was diluted 100 times with gold-labeling diluent and then mixed with the equine infectious anemia virus p26-gp90 fusion protein colloidal gold complex diluted in step 1.2. The mixture was then soaked in glass fiber and dried at 37°C for 4 hours to prepare the gold-labeled pad.

[0085] 1.4 Dotted membrane formation of recombinant equine infectious anemia virus p26-gp90 fusion protein

[0086] The purified p26-gp90 fusion protein was diluted to 0.9 mg / mL with spot dilution buffer (50 mM Tris, 2% sucrose, pH 8.5) to serve as the test line (T line) for the colloidal gold test strip. Goat anti-mouse IgG (Hangzhou LONGi Biotechnology Co., Ltd., catalog number: PS00901) was diluted to 0.3 mg / mL with the same dilution buffer to serve as the control line (C line) for the colloidal gold test strip. The two diluted solutions were then streaked onto a nitrocellulose membrane and dried at 37°C overnight.

[0087] 1.5 Assembly of a test strip for detecting equine infectious anemia virus antibodies using a double-antigen sandwich gold labeling method

[0088] The gold-labeled pad, the polyester plate coated with the raw materials onto the nitrocellulose membrane (NC membrane), filter paper, sample pad, etc., are installed on the base plate to assemble the equine infectious anemia virus antibody double antigen sandwich test strip. Specific installation method is as follows: Figure 2 As shown: Sample pad 1, gold label pad 2, NC membrane 3, and filter paper 4 are respectively installed on base plate 5. A portion of sample pad 1 is stacked on top of gold label pad 2, a portion of gold label pad 2 is stacked on top of NC membrane 3, and a portion of filter paper 4 is stacked on top of NC membrane 3. NC membrane 3 is divided into a testing area and a quality control area. The testing area has a detection line 31 (T line), and the quality control area has a quality control line 32 (C line). Detection line 31 is close to gold label pad 2, and quality control line 32 is close to filter paper 4.

[0089] Furthermore, the assembled test strips are cut into 3mm strips using a strip cutter, and then inserted into specially designed plastic cards to become a complete test reagent card.

[0090] 2. Double-antigen sandwich gold label method for detecting equine infectious anemia virus antibody test strips / cards

[0091] Add 90 μL of the sample to be tested (horse serum, plasma) to the sample loading point (S), incubate at room temperature for 10 minutes, and then determine the results. The result determination criteria are as follows (e.g., Figure 3 As shown):

[0092] ④ The presence of two bands, one in the control area and the other in the test area, indicates a positive result;

[0093] ⑤ If only one band appears on the control line and no band appears in the test area, the result is negative;

[0094] ⑥ If no band appears on the control line, it indicates that the test strip is damaged. Regardless of whether a band appears on the test line, a new test strip should be used and the test repeated.

[0095] Detection results of 3-antigen sandwich gold label method for detecting equine infectious anemia virus antibody test strips / cards

[0096] A total of 20 positive equine infectious anemia virus (EIAV) serum samples (sample numbers 1-20) and 50 normal, disease-free, and unimmunized equine serum samples (sample numbers 21-70) were tested. Two lines, T and C, indicate a positive result, while only one line, C, indicates a negative result.

[0097] The test results are shown in Table 1: 19 positive cases were detected in 20 positive serum samples, and 1 case was missed (sample 16). 3 false positives were found in 50 negative serum samples (samples 26, 34 and 41).

[0098] Table 1 Results of equine infectious anemia virus antibody detection

[0099]

[0100]

[0101] Therefore, the sensitivity and specificity of the sample detection are 99.5% and 94%, respectively, with an overall concordance rate of 94.3%. Figure 4 As shown.

[0102] The above results demonstrate that the recombinant equine infectious anemia virus p26-gp90 fusion protein of the present invention has very high sensitivity and specificity for detecting equine infectious anemia virus. It can be used as a raw material for making equine infectious anemia virus antibody test strips and can be widely used in clinical testing.

[0103] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A kit for detecting antibodies to equine infectious anemia virus, characterized in that, The kit includes a double-antigen sandwich gold labeling test strip, and the test strip is prepared as follows: S1, prepare colloidal gold complex of equine infectious anemia virus p26-gp90 recombinant protein and mouse IgG colloidal gold complex, respectively, wherein the equine infectious anemia virus p26-gp90 recombinant protein is composed of the amino acid sequence shown in SEQ ID NO. 1; S2, the equine infectious anemia virus p26-gp90 recombinant protein colloidal gold complex and mouse IgG colloidal gold complex are mixed to prepare a gold-labeled pad; S3, using the recombinant protein of equine infectious anemia virus p26-gp90 as the detection line and sheep anti-mouse IgG as the quality control line, was drawn on a nitrocellulose membrane; S4. Filter paper, a polyester plate containing a nitrocellulose membrane, a gold label pad, and a sample pad are mounted on a base plate. A portion of the filter paper is stacked on the polyester plate, a portion of the polyester plate is stacked on the gold label pad, and a portion of the gold label pad is stacked on the sample pad. The polyester plate has a test area and a quality control area. The test area has a detection line, and the quality control area has a quality control line. The detection line is close to the gold label pad, and the quality control line is close to the filter paper, thus preparing the test strip.

2. The kit of claim 1, wherein The preparation method of the equine infectious anemia virus p26-gp90 recombinant protein colloidal gold complex is as follows: Take 100 mL of colloidal gold solution and put it into a beaker. Add 0.2 M K2CO3 to adjust the pH of the gold solution to 6.

5. After stirring, add 1.5 mg of purified equine infectious anemia virus p26-gp90 recombinant protein. Stir at room temperature for 15 min, add 1 mL of 10% BSA solution, stir at room temperature for 15 min, centrifuge at 12000 rpm for 10 min, carefully aspirate and discard the supernatant, and make up the volume of the precipitate to 1 mL with gold standard diluent, which includes 20 mM Tris, 1% BSA, 0.03% Proclin 300, and pH 8.

0.

3. The kit of claim 2, wherein The colloidal gold solution is prepared as follows: Add 1000 mL of ultrapure water to an Erlenmeyer flask and heat to boiling on a magnetic stirrer. Then add 4 mL of 10% chloroauric acid and 6 mL of 10% trisodium citrate solution. Continue heating to boiling for 5 min. After cooling to room temperature, filter through a 0.22 μm filter and store at 4°C for later use.

4. The kit of claim 2, wherein The purification process involves passing the recombinant equine infectious anemia virus p26-gp90 protein through a Ni column and eluting it with an elution buffer comprising 50 mM Tris, 0.2 M NAcl, 0.5 M Imidazole, and pH 8.

0.

5. The kit of claim 2, wherein The preparation method of the mouse IgG colloidal gold complex is as follows: Take 100 mL of colloidal gold solution and put it into a beaker. Add 0.2 M K2CO3 and adjust the pH of the gold solution to 6.

5. After stirring, add 1 mg of mouse IgG and stir at room temperature for 15 min. Add 1 mL of 10% BSA solution and stir at room temperature for 15 min. Centrifuge at 12000 rpm for 10 min. Carefully aspirate and discard the supernatant. Dilute the precipitate to 1 mL with gold standard diluent, which includes 20 mM Tris, 1% BSA, 0.03% Proclin 300, and pH 8.

0.

6. The kit of claim 5, wherein The mouse IgG colloidal gold complex was diluted 100 times with gold label diluent and then mixed with the equine infectious anemia virus p26-gp90 recombinant protein colloidal gold complex. The mixture was then soaked in glass fiber and dried at 37°C for 4 hours to prepare the gold label pad.

7. The kit of claim 1, wherein The purified equine infectious anemia virus p26-gp90 recombinant protein was diluted to 0.9 mg / mL using spot dilution buffer and used as the detection line for the colloidal gold test strip. Goat anti-mouse IgG was diluted to 0.3 mg / mL using the same dilution buffer and used as the control line for the colloidal gold test strip. The two diluted solutions were then streaked onto a nitrocellulose membrane and dried at 37°C overnight.

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