A kit for detecting ehrlichia canis antibodies

Abstract

The application discloses a kit for detecting antibodies of canine ehrlichia, and belongs to the field of animal virus antibody detection.The kit comprises canine ehrlichia MAP2-P30-gp19 recombinant protein, mouse IgG and goat anti-mouse IgG, and the MAP2-P30-gp19 recombinant protein is composed of an amino acid sequence shown in SEQ ID NO.1.The kit is used for detecting the antibodies of canine ehrlichia, and has the advantages of convenience, high sensitivity, no cross reaction with other pathogens, high specificity, great clinical significance and wide application prospect.

Description

[0001] Related Patents

[0002] The present application is a divisional application of the Chinese Invention Patent Application No. 2020114758176, filed on December 14, 2020, entitled "A Canine Ehrlichia MAP2-P30-gp19 Recombinant Protein and Preparation Method and Application Thereof". TECHNICAL FIELD

[0003] The present application belongs to the field of animal virus antibody detection, and specifically relates to a kit for detecting canine Ehrlichia antibody. BACKGROUND

[0004] Canine Ehrlichiosis is a canine septicemic infectious disease caused by Ehrlichia canis transmitted by cattle ticks. Domestic dogs, wild dogs and rodents are the hosts of the disease. Dogs of various genders, ages and breeds can be infected with the disease. The incubation period of the disease is 8-12 days. The sick dog has periodic fever, anorexia, mucous and purulent nasal fluid and eye discharge, anemia and weight loss.

[0005] The more direct diagnosis method in the clinic at present is blood smear staining method, indirect fluorescence antibody test (Indirect fluorescence antibody, IFA) and enzyme-linked immunosorbent assay (Enzyme-Linked immunosorbent assay, ELISA) and other immunological methods, in addition, PCR (polymerase chain reaction) method is also a common method for Ehrlich diagnosis, which is the most sensitive and specific detection method for diagnosing Ehrlich disease. However, the above-mentioned immunofluorescence method, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) technology and other methods need to use specified instruments and equipment, have corresponding test conditions and skills, and are difficult to popularize in the grass-roots level.

[0006] In terms of Ehrlichia antibody detection, the main direction of the current domestic and foreign research is to use one or several partial sequences to detect antibodies, which inevitably has the risk of missed detection. SUMMARY

[0007] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0008] The first aspect of the present application provides a canine Ehrlichia MAP2-P30-gp19 recombinant protein comprising the amino acid sequence shown in SEQ ID NO. 1.

[0009] In the present application, the recombinant protein is also called fusion protein or recombinant fusion protein, which is the expression product of two genes recombined by DNA recombination technology.

[0010] The proteins such as MAP2 (Major Antigenic Protein 2), outer membrane protein (Major Outer Membrane Protein) such as P30, P30a, P30-1 and glycoprotein (Glycoprotein) such as gp19 are the main immunogenic proteins of canine Ehrlichia, which can induce and initiate the immune response of the body immune system. The full-length sequences of MAP2, P30 and gp19 proteins are fused and expressed in the present application, which can not only improve the sensitivity of diagnosis, but also reduce the cross-reaction with other pathogens.

[0011] In some embodiments of the present application, preferably, the recombinant protein consists of the amino acid sequence shown in SEQ ID NO. 1.

[0012] The second aspect of the present application provides a gene encoding the recombinant protein of the first aspect of the present application, which comprises the nucleotide sequence shown in SEQ ID NO. 2.

[0013] The gene sequence is for expressing the recombinant protein in E. coli, and the codons are optimized according to the preference of E. coli for codons. The frequency of using synonymous codons is different in different species, and this codon preference has an impact on the translation process. If an mRNA has many clustered rare codons, this will negatively affect the speed of ribosome movement, greatly reducing the protein expression level. The gene sequence is codon-optimized in this application, which is suitable for E. coli expression and can improve the protein expression efficiency.

[0014] The third aspect of the present application provides an expression vector comprising the gene of the second aspect of the present application.

[0015] In some embodiments of the present application, the expression vector is pET30a, which has kanamycin resistance, and the expressed fusion protein has a histidine (His) tag.

[0016] The fourth aspect of the present application provides a host cell containing the expression vector of the third aspect of the present application.

[0017] Further, the host cell is a eukaryotic host cell or a prokaryotic host cell.

[0018] In some embodiments of the present application, the host cell is a prokaryotic host cell. Preferably, the host cell is E. coli, and more preferably, the E. coli is BL21. Using E. coli for expression has the advantages of short cycle, low cost and large expression amount.

[0019] The fifth aspect of the present application provides a method for preparing the recombinant protein of the first aspect of the present application, comprising the step of inducing the host cell of the fourth aspect of the present application to express the protein.

[0020] Further, the host cell is a eukaryotic host cell or a prokaryotic host cell.

[0021] In some embodiments of the present application, the host cell is a prokaryotic host cell. Preferably, the host cell is Escherichia coli, more preferably, the Escherichia coli is BL21. Using Escherichia coli for expression has the advantages of short cycle, low cost, large expression amount, etc.

[0022] In some specific embodiments of the present application, the step of inducing the Escherichia coli to express the protein is:

[0023] S1, culturing the Escherichia coli in LB medium containing 50 μg / mL kanamycin at 37°C,

[0024] S2, when the OD600 of the Escherichia coli culture solution is 0.5-0.7, inducing expression with IPTG at a final concentration of 1 mM, the induction conditions are: 25°C, 200 rpm, 4h; using this induction condition, the recombinant protein is expressed more slowly, and there is sufficient time for the formation of spatial conformation, which is very important for the recombinant protein to function.

[0025] S3, centrifuging the culture solution at 4°C, 7000 rpm for 10 min to collect the bacterial cells;

[0026] S4, breaking the bacterial cells with buffer Binding Buffer;

[0027] S5, ultrasonic breaking of the bacterial cells, the conditions are: 500w, ultrasonic for 2s, interval 5s, a total of 80-120 times;

[0028] S6, centrifuging at 4°C, 12000 rpm for 30 min to collect the supernatant, and the recombinant protein is in the supernatant.

[0029] Preferably, the induction in step S2 is performed when the OD600 of the Escherichia coli culture solution is 0.6.

[0030] Preferably, in step S5, the ultrasonic breaking is performed 100 times. Using the breaking method of the present application, the situation of excessive breaking of the recombinant protein is avoided.

[0031] In some embodiments of the present application, a step of purifying the recombinant protein is further included. The recombinant protein can be purified by various methods, such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography. In some embodiments of the present application, the method of affinity chromatography is selected, because the recombinant protein has a His tag, and one-step purification can achieve a high purity.

[0032] In some specific embodiments of the present application, the supernatant containing the recombinant protein is passed through a Ni column, and then eluted with an elution buffer to obtain the target protein.

[0033] Preferably, the elution buffer has a formula of 50 mM Tris, 0.2 M Nacl, 0.5 M Imidazole, pH 8.0.

[0034] The sixth aspect of the present application provides the use of the recombinant protein of the first aspect of the present application in the preparation of a kit for detecting antibodies against Ehrlichia canis.

[0035] The seventh aspect of the present application provides a kit for detecting antibodies against Ehrlichia canis, comprising the recombinant protein of the first aspect of the present application.

[0036] Further, the kit further comprises mouse IgG and goat anti-mouse IgG.

[0037] In some embodiments of the present application, the antibodies against Ehrlichia canis are detected by a double antigen sandwich gold label method.

[0038] In some specific embodiments of the present application, the kit comprises a double antigen sandwich gold label detection strip, and the reagent method of the detection strip is as follows:

[0039] S1, preparing a recombinant protein colloidal gold complex and a mouse IgG colloidal gold complex, respectively;

[0040] S2, mixing the recombinant protein colloidal gold complex and the mouse IgG colloidal gold complex to prepare a gold label pad;

[0041] S3, drawing on a nitrocellulose membrane using the recombinant protein as a detection line and using goat anti-mouse IgG as a quality control line;

[0042] S4, mounting filter paper, a polyester plate containing the nitrocellulose membrane, a gold label pad, and a sample pad on a base plate, wherein a portion of the filter paper is stacked and pressed on the polyester plate, a portion of the polyester plate is stacked and pressed on the gold label pad, and a portion of the gold label pad is stacked and pressed on the sample pad, the polyester plate has a test area and a quality control area, respectively, 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, thereby obtaining a detection strip.

[0043] In use, the dog biological sample is added dropwise to the sample pad, and the detection result is determined after standing at room temperature for 10 min, and the determination criteria are as follows:

[0044] ①Two bands appear, one of which is located in the quality control zone, and the other is located in the test zone, which is positive;

[0045] ②Only one band appears in the quality control line, and no band appears in the test zone, which is negative;

[0046] ③The quality control line does not appear a band, indicating that the test strip is damaged, and whether the detection line appears a band or not, a new test strip should be replaced for retesting.

[0047] In some embodiments of the present application, the positive result of the detection of E. canis antibodies indicates that the dog biological sample contains E. canis antibodies, which means that the dog has been infected with E. canis or has been infected with E. canis.

[0048] In some embodiments of the present application, the biological sample is serum or plasma, or any other body fluid that may contain antibodies.

[0049] Advantages of the present application

[0050] Compared with the prior art, the present application has the following advantages:

[0051] MAP2, P30 and gp19 proteins are the main immunogenic antigens of E. canis and are highly conserved, which can induce and initiate the immune system of the body to produce an immune response and induce host cells to produce neutralizing antibodies. Therefore, the fusion of the full-length sequences of MAP2, P30 and gp19 proteins not only improves the sensitivity of diagnosis, but also reduces cross-reactions with other pathogens, has strong specificity, and has great clinical significance and wide application prospects.

[0052] The colloidal gold-labeled immunoassay used in the present application is a new type of analysis technology, which is fast, simple, low-cost, non-polluting and does not require training. Compared with traditional methods, it is more suitable for on-site detection, has the advantages of short color development time and does not require expensive instruments, and has broad market prospects and application value. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 The gel electrophoresis results of the purification of the E. canis MAP2-P30-gp19 fusion protein are shown. 1: cell disruption; 2: flow-through; 3: 50mM Imidazole elution; 4: 0.5M Imidazole elution.

[0054] Figure 2Reagent diagram of test strip of one embodiment of the present application. 1: sample pad; 2: gold pad; 3: NC membrane; 31: detection line (T line); 32: quality control line (C line); 4: filter paper; 5: bottom plate.

[0055] Figure 3 The result diagram of detection using test strip of one embodiment of the present application is shown. T: detection line, C: quality control line.

[0056] Figure 4 The overall result of detection of clinical canine serum samples using test strip of the present application is shown. DETAILED DESCRIPTION

[0057] In order to make the technical problems solved by the present application, technical solutions and beneficial effects more clear, the present application is further described in detail below in combination with embodiments.

[0058] EMBODIMENTS

[0059] The following examples are presented to demonstrate preferred embodiments of the present application. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques that the inventors have found to function well in the practice of the present application and thus can be considered to be preferred embodiments of the present application. However, those skilled in the art will further appreciate that many of the techniques disclosed in the following examples can be easily modified or altered to function in the practice of the present application without departing from the spirit or scope of the present application.

[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 application belongs. The materials, methods, and examples provided herein are illustrative only and are not intended to be limiting.

[0061] One skilled in the art will appreciate that many of the specific embodiments of the application described herein can be made using alternative techniques and materials without departing from the spirit or scope of the application. Such alternatives are considered to be within the scope of the present application.

[0062] The experimental methods in the following examples are all 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 test materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0063] Example 1 Construction of canine ehrlichia MAP2-P30-gp19 fusion protein gene expression vector

[0064] The canine Ehrlichia MAP2 gene is designed according to the protein sequence of NCBI Gene bank: AF117731, the P30 gene is designed according to the protein sequence of NCBI Gene bank: WP_011305021.1, and the gp19 gene is designed according to the protein sequence of NCBI Gene bank: ABU44523.

[0065] The amino acid sequence of the MAP2-P30-gp19 recombinant fusion protein is as follows (SEQ ID NO. 1):

[0066] MKVIKFILNICLLFAAIFLGYSYVTKQGIFQVRDHNTPNTNISNKASITTSFSLVNQDGNTVNSQDFLGKYMLVLFGFSSCKSICPAELGIASEVLSQLGNDTDKLQVIFITIDPTNDTVQKLKTFHEHFDPRIQMLTGSAEDIEKIIKNYKIYVGQADKDNQIDHSAIMYIIDKKGEYISHFSPDLKSTENQVDKLLSIIKQYLMNCKRFFIASALISLMSFLPSVSFSESIHEDNINGNFYISAKYMPSASHFGVFSVKEEKNTTTGVFGLKQDWDGATIKDASSSHTIDPSTIFSISNYSFKYENNPFLGFAGAIGYSMGGPRVEFEVSYEIFDVKNQGNSYKNDAHKYCALSRHTGGMPQAGHQNKFVFLKNEGLLDISLMINACYDITIDSMPFSPYICAGIGSDLVSMFETTNPKISYQGKLGVSYSISPEASVFVGGHFHRVIGNEFKDIPAITPAGATEIKGTQFTTVTLNICHFGLELGGRFTFMLQVQNHVDQHTNHIEHDDYHFTGPTSFEVNLSEEEKMELQEVSSIDSVGCEDCDPNCRYPLELVECQRIEERPVCNAGLESLTVDAYQLGLLLGGFLSAMNYISYSCPCYYYDCCDRNYYDCYHKNACYYNCCDCA

[0067] Different species have different frequencies of synonymous codon usage, and this codon bias has an impact on the translation process. If an mRNA has many clustered rare codons, this will negatively affect the speed of ribosome movement, greatly reducing protein expression levels.

[0068] The present application uses E. coli as an expression system, in order to obtain higher expression efficiency and higher expression amount, codon optimization is carried out when expressing exogenous proteins, and the obtained nucleotide sequence is as follows (SEQ ID NO. 2):

[0069]

[0070] The recombinant gene sequence was synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. and linked with pET30a plasmid to form a recombinant expression vector.

[0071] Example 2 Expression of E. canis MAP2-P30-gp19 fusion protein

[0072] The E. canis MAP2-P30-gp19 fusion gene plasmid was transformed into E. coli BL21 and plated on LB plates containing 50 μg / mL kanamycin (Shanghai Shenguo, Catalog No.: K0408) and incubated at 37°C overnight. Single colonies were picked and cultured in 300 mL LB medium containing the same concentration of kanamycin at 37°C until the OD600 reached about 0.6. IPTG (Shanghai Shenguo, Catalog No.: IB0168) was used for induction at a final concentration of 1 mM. The induction conditions were 25°C, 200 rpm for 4 h. After induction, the culture was centrifuged at 4°C and 7000 rpm for 10 min to collect the bacterial cells.

[0073] Example 3 Purification and renaturation of E. canis MAP2-P30-gp19 fusion protein

[0074] The bacterial cells were disrupted by 50 mL of Binding Buffer (50 mM Tris, 0.2 M Nacl, pH 8.0). Then the cells were disrupted by ultrasonic treatment at 500 w for 2 s with an interval of 5 s for a total of 100 times. Finally, the supernatant was collected by centrifugation at 12000 rpm for 30 min at 4°C. The target protein was in the supernatant. Then the Ni column was used for one-step purification, and the target protein was eluted 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 in Figure 1 .

[0075] From Figure 1 It can be seen that the purity of the purified fusion protein is very high. The purified recombinant protein was dialyzed with dialysis buffer (50 mM Tris, 0.2 M Nacl, pH 8.0) every 12 h for a total of 3 times. The dialyzed protein solution was filtered through a 0.22 μm filter, and the concentration was determined by BCA method. The solution was stored at -20°C for future use.

[0076] Example 4 Detection of E. canis antibodies by double antigen sandwich gold labeling method

[0077] 1 Preparation of double antigen sandwich gold labeling method detection strip

[0078] 1.1 Preparation of colloidal gold

[0079] In a triangular flask, 1000 mL of ultrapure water was added, heated to boiling on a magnetic heating stirrer, then 4 mL of 10% chloroauric acid (sigma) was added, then 6 mL of 10% trisodium citrate solution was added, and boiling was continued for 5 min, then cooled to room temperature, and then filtered with a 0.22 μm filter after colloidal gold was placed at 4°C for standby.

[0080] 1.2 Labeling of recombinant canine ehrlichia MAP2-P30-gp19 fusion protein

[0081] Take 100 mL of colloidal gold solution into a beaker, stir and add 0.2M K2CO3 to adjust the pH of the gold water to 6.5, then add 1.2 mg of purified recombinant canine ehrlichia MAP2-P30-gp19 fusion protein after stirring, stir at room temperature for 15 min, add 1 mL of 10% BSA solution, stir at room temperature for 15 min, then centrifuge at 12000 rpm for 10 min, carefully aspirate and discard the supernatant, and dilute the precipitate to 1 mL with gold labeling diluent (20 mM Tris, 1% BSA, 0.03% Proclin300, pH 8.0), which is the labeled recombinant canine ehrlichia MAP2-P30-gp19 fusion protein colloidal gold complex.

[0082] 1.3 Mouse IgG labeling

[0083] Take 100 mL of colloidal gold solution into a beaker, stir and add 0.2M K2CO3 to adjust the pH of the gold water to 7.0, then add 1 mg of mouse IgG (Hangzhou Longke Biotechnology Co., Ltd., Item No.: AS00901) after stirring, stir at room temperature for 15 min, add 1 mL of 10% BSA solution, stir at room temperature for 15 min, then centrifuge at 12000 rpm for 10 min, carefully aspirate and discard the supernatant, and dilute the precipitate to 1 mL with gold labeling diluent (20 mM Tris, 1% BSA, 0.03% Proclin300, pH 8.0), which is the labeled mouse IgG colloidal gold complex.

[0084] Dilute the above gold labeling complex 100 times with gold labeling diluent, then mix with the diluted canine ehrlichia MAP2-P30-gp19 fusion protein colloidal gold complex in step 1.2, then soak the glass fiber, dry at 37°C for 4 h, and then prepare the gold labeling pad.

[0085] 1.4 Dotting of recombinant canine ehrlichia MAP2-P30-gp19 fusion protein

[0086] The purified MAP2-P30-gp19 fusion protein was diluted to 0.7 mg / mL with a dot membrane diluent (50 mM Tris, 2% sucrose, pH 8.5) as the test line (T line) of the colloidal gold test strip, and the goat anti-mouse IgG (Hangzhou Longke Biotechnology Co., Ltd., Item No.: PS00901) was diluted to 0.3 mg / mL with the same diluent as the control line (C line) of the colloidal gold test strip. The above two diluted solutions were drawn on a nitrocellulose membrane, and dried at 37°C overnight.

[0087] 1.5 Assembly of the double antigen sandwich gold label method for detecting canine ehrlichia antibody test strip

[0088] The above gold label pad, polyester plate and filter paper coated with raw materials on the nitrocellulose membrane (NC membrane), sample pad, etc. were installed on the base plate to assemble the double antigen sandwich method for detecting canine ehrlichia antibody test strip. The specific installation method is shown in Figure 2 The sample pad 1, gold label pad 2, NC membrane 3 and filter paper 4 were installed on the base plate 5, respectively. The sample pad 1 was stacked on a part of the gold label pad 2, the gold label pad 2 was stacked on a part of the NC membrane 3, and the filter paper 4 was stacked on a part of the NC membrane 3. The NC membrane 3 was divided into a test area and a control area, the test line 31 (T line) was set in the test area, and the control line 32 (C line) was set in the control area. The test line 31 was close to the gold label pad 2, and the control line 32 was close to the filter paper 4.

[0089] Further, the assembled test strip was cut into 3 mm strips with a strip cutter, and then was loaded into a specially designed plastic card, which became a mature detection reagent card.

[0090] 2 Detection of the double antigen sandwich gold label method for detecting canine ehrlichia antibody test strip / card

[0091] 90 μL of the sample to be detected (canine serum, plasma) was added to the sample addition site (S), and the result was determined after standing at room temperature for 10 min. The result determination criteria are as follows (as shown in Figure 3 ).

[0092] ④ Two bands appeared, one in the control area and the other in the test area, which was positive;

[0093] ⑤ Only the control line appeared a band, and no band appeared in the test area, which was negative;

[0094] ⑥ The control line did not appear a band, indicating that the test strip was damaged, and whether the test line appeared a band or not, a new test strip should be replaced for retesting.

[0095] 3 Detection results of the double antigen sandwich gold label method for detecting canine ehrlichia antibody test strip / card

[0096] 25 samples of canine Ehrlichia infection positive serum (sample No. 1-25) and 49 samples of normal non-disease and non-immune serum (sample No. 26-74) were detected, wherein the detection results of T line and C line were positive, and the detection result of only C line was negative.

[0097] The detection results are shown in Table 1: 20 of 25 positive serum samples were detected positive, 0 was missed, and 1 of 49 negative serum samples was false positive (sample No. 68).

[0098] Table 1: Detection results of canine Ehrlichia antibody

[0099]

[0100]

[0101]

[0102] Therefore, the sensitivity and specificity of sample detection are 100% and 97.6%, respectively, and the overall coincidence rate is 98.6%, as shown in Table 2. Figure 4

[0103] The above results show that the recombinant canine Ehrlichia MAP2-P30-gp19 fusion protein of the application has very high sensitivity and specificity in detecting canine Ehrlichia, can be used as raw material for preparing canine Ehrlichia antibody detection test strips, and can be widely used in clinical detection.

[0104] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the appended claims of the present application.​

Claims

1. A kit for detecting antibodies to Ehrlichia canis, characterized in that, The MAP2-P30-gp19 recombinant protein of canine ehrlichia, mouse IgG and goat anti-mouse IgG are included, and the MAP2-P30-gp19 recombinant protein consists of the amino acid sequence shown in SEQ ID NO.

1.

2. The kit of claim 1, wherein The kit uses a double antigen sandwich gold mark method to detect canine ehrlichia antibodies.

3. The kit of claim 2, wherein The kit includes a double antigen sandwich gold mark detection strip, and the preparation method of the detection strip is as follows: S1, respectively prepare MAP2-P30-gp19 recombinant protein colloidal gold complex and mouse IgG colloidal gold complex; S2, mix the MAP2-P30-gp19 recombinant protein colloidal gold complex and the mouse IgG colloidal gold complex to prepare a gold mark pad; S3, draw on the nitrocellulose membrane using MAP2-P30-gp19 recombinant protein as the detection line and goat anti-mouse IgG as the quality control line; S4, install the filter paper, the polyester plate containing the nitrocellulose membrane, the gold mark pad and the sample pad on the bottom plate, wherein a part of the filter paper is stacked and pressed on the polyester plate, a part of the polyester plate is stacked and pressed on the gold mark pad, and a part of the gold mark pad is stacked and pressed on the sample pad, the test area and the quality control area are respectively provided on the polyester plate, the test area has the detection line, and the quality control area has the quality control line, the detection line is close to the gold mark pad, and the quality control line is close to the filter paper, that is, the detection strip is prepared.

4. The kit of claim 3, wherein The preparation method of the MAP2-P30-gp19 recombinant protein colloidal gold complex is as follows: Take 100 mL of colloidal gold solution into a beaker, stir and add 0.2M K2CO3 to adjust the pH of the gold water to 6.5, then stir and add 1.2 mg of purified MAP2-P30-gp19 recombinant protein, stir at room temperature for 15 min, add 1 mL of 10% BSA solution, stir at room temperature for 15 min, then centrifuge at 12000 rpm for 10 min, carefully aspirate and discard the supernatant, and dilute the precipitate to 1 mL with a gold mark diluent, which includes 20 mM Tris, 1% BSA, 0.03% Proclin 300, pH 8.

0.

5. The kit of claim 4, wherein The preparation method of the colloidal gold solution is as follows: Add 1000 mL of ultrapure water to a triangular flask, heat to boiling on a magnetic heating stirrer, then add 4 mL of 10% chloroauric acid and 6 mL of 10% trisodium citrate solution, continue to heat to boiling for 5 min, then cool to room temperature, filter with a 0.22 μm filter and store at 4°C for standby.

6. The kit of claim 4, wherein The purification refers to passing the MAP2-P30-gp19 recombinant protein through a Ni column, and eluting with an elution buffer, which includes 50 mM Tris, 0.2 M Nacl, 0.5 M Imidazole, pH 8.

0.

7. The kit of claim 4, wherein The preparation method of the mouse IgG colloidal gold complex is as follows: Take 100 mL colloidal gold solution into a beaker, add 0.2 M K2CO3 to adjust the pH of the gold solution to 6.5, then add 1 mg mouse IgG after stirring, stir at room temperature for 15 min, add 1 mL 10% BSA solution, stir at room temperature for 15 min, then centrifuge at 12000 rpm for 10 min, carefully aspirate and discard the supernatant, and dilute the precipitate to 1 mL with gold label diluent, which comprises 20 mM Tris, 1% BSA, 0.03% Proclin 300, pH 8.

0.

8. The kit of claim 7, wherein After diluting the mouse IgG colloidal gold complex 100 times with the gold label diluent, mix it with the MAP2-P30-gp19 recombinant protein colloidal gold complex, then soak the glass fiber, dry at 37°C for 4 h, and thus prepare the gold label pad.

9. The kit of claim 3, wherein Dilute the purified MAP2-P30-gp19 recombinant protein to 0.7 mg / mL with dot membrane diluent to serve as the detection line of the colloidal gold test strip, dilute goat anti-mouse IgG to 0.3 mg / mL with the same diluent to serve as the quality control line of the colloidal gold test strip, draw the above two diluted solutions onto the nitrocellulose membrane, dry at 37°C overnight, and the dot membrane diluent comprises 50 mM Tris, 2% sucrose, pH 8.5.

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