Toxoplasma gondii chimeric antigen and application thereof in detection kit

Abstract

The invention provides a toxoplasma gondii chimeric antigen and application thereof in preparation of a kit for detecting toxoplasmosis, the chimeric antigen is composed of a plurality of epitopes, and the sequence of the chimeric antigen is shown as SEQ ID No.8. When the toxoplasma gondii chimeric antigen is used as a diagnosis antigen of toxoplasmosis, especially a gold-labeled antigen of a colloidal gold immunochromatography test strip for detecting the toxoplasmosis, the toxoplasmosis can be rapidly, accurately and conveniently detected, and the toxoplasma gondii chimeric antigen has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, and in particular to a Toxoplasma gondii chimeric antigen and application thereof in a kit for detecting toxoplasmosis. Background Art

[0002] Specific drugs for the treatment of toxoplasmosis have not yet been successfully developed, so early diagnosis, early warning, and early prevention have become important means to control the spread of toxoplasmosis. Currently, the diagnostic methods for toxoplasmosis include a variety of methods such as etiology, serology, and molecular biology detection. These methods have their own advantages and disadvantages due to different operating steps and required equipment. For example, although etiology detection is simple to operate, its accuracy and detection rate are low and cannot meet the needs of modern efficient detection. Agglutination tests, such as the modified agglutination test (MAT), are prone to false negative results. Molecular biology testing, due to the complexity of serum components, the results are unstable.

[0003] Immunochromatographic test (ICT) is a rapid detection technology that is becoming more and more common. It uses cellulose membrane as a carrier and utilizes the principle of antigen-antibody specific reaction and the traceability of colloidal gold particles for detection. The present invention uses recombinant multi-epitope chimeric antigen as a diagnostic antigen to develop a colloidal gold immunochromatographic test strip for toxoplasmosis. Summary of the invention

[0004] The invention provides a Toxoplasma gondii chimeric antigen and application thereof in preparing a kit for detecting toxoplasmosis.

[0005] On the one hand, the present invention provides a Toxoplasma gondii chimeric antigen, which includes antigenic epitopes of surface antigen 1 (Surface Antigen 1), surface antigen 3 (Surface Antigen 3), bradyzoite antigen 1 (BradyzoiteAntigen 1), rhoptry protein 2 (Rhoptry Protein 2) and dense granule protein 54 (Dense GranuleProtein 54).

[0006] In one embodiment, the chimeric antigen further comprises a linker. In one embodiment, the sequence of the linker is shown in SEQ ID No.7.

[0007] In one embodiment, the chimeric antigen includes antigenic epitopes of surface antigen 1 (Surface Antigen 1), surface antigen 3 (Surface Antigen 3), bradyzoite antigen 1 (Bradyzoite Antigen 1), rhoptry protein 2 (Rhoptry Protein 2) and dense granule protein 54 (Dense Granule Protein 54), and the sequences of the antigenic epitopes are shown in SEQ ID No.1-6.

[0008] In one embodiment, the chimeric antigen comprises the sequence shown in SEQ ID No. 1-7.

[0009] In one embodiment, the chimeric antigen is composed of an antigenic epitope of surface antigen 1 (Surface Antigen 1) shown in SEQ ID No.1, a linker shown in SEQ ID No.7, an antigenic epitope of surface antigen 3 (Surface Antigen 3) shown in SEQ ID No.2, a linker shown in SEQ ID No.7, an antigenic epitope of bradyzoite antigen 1 (Bradyzoite Antigen 1) shown in SEQ ID No.3, a linker shown in SEQ ID No.7, an antigenic epitope of rhoptry protein 2 (Rhoptry Protein 2) shown in SEQ ID No.4, a linker shown in SEQ ID No.7, an antigenic epitope of dense granule protein 54 (Dense Granule Protein 54) shown in SEQ ID No.5, a linker shown in SEQ ID No.7, and an antigenic epitope of surface antigen 1 (Surface Antigen 1) shown in SEQ ID No.6, which are connected in sequence.

[0010] In one embodiment, the sequence of the chimeric antigen is shown as SEQ ID No.8.

[0011] In one embodiment, the chimeric antigen is obtained by prokaryotic expression.

[0012] In one embodiment, the chimeric antigen is obtained by prokaryotic expression and purification.

[0013] On the other hand, the present invention provides the use of the chimeric antigen in preparing a toxoplasmosis detection kit.

[0014] In one embodiment, the toxoplasmosis detection kit is a colloidal gold immunochromatographic test strip.

[0015] On the other hand, the present invention provides a colloidal gold immunochromatographic test strip for detecting toxoplasmosis, wherein the test strip uses the chimeric antigen as a gold-labeled antigen.

[0016] In one embodiment, the test strip comprises a reaction membrane, a sample pad, a gold label pad, a water absorbent pad and a backing, and the gold label pad is coated with the colloidal gold label of the chimeric antigen.

[0017] In one embodiment, the reaction membrane is coated with a quality control area of ​​an antibody that can bind to the chimeric antigen.

[0018] In one embodiment, the antibody is a polyclonal antibody.

[0019] In one embodiment, the antibody is prepared using an immune response of an animal, and the animal is a rabbit.

[0020] In one embodiment, the antibody is prepared by introducing the chimeric antigen into an animal and utilizing the immune response of the animal.

[0021] In one embodiment, the reaction membrane has a test area coated with recombinant protein A.

[0022] In one embodiment, the reaction membrane is a NC membrane (nitrocellulose membrane).

[0023] In one embodiment, the test strip further comprises a loading cartridge.

[0024] In another aspect, the present invention provides a method for preparing a colloidal gold immunochromatographic test strip for toxoplasmosis, the method comprising the steps of:

[0025] 1) Expression and purification of the chimeric antigen;

[0026] 2) preparing a gold label pad, on which the colloidal gold label of the chimeric antigen is coated;

[0027] 3) preparing a reaction membrane comprising a test area coated with recombinant protein A and a quality control area coated with an antibody capable of binding to the chimeric antigen;

[0028] 4) Assembling the prepared gold label pad, reaction membrane, sample pad, water absorbent pad and backing into the test strip.

[0029] In one embodiment, the colloidal gold marker of the chimeric antigen is prepared by the following steps:

[0030] (a) preparing a colloidal gold solution; preferably, the colloidal gold is prepared by reacting chloroauric acid and sodium citrate solution in boiling water.

[0031] (b) preparing colloidal gold recombinant protein, adding a buffer to the colloidal gold solution to adjust the pH, adding the chimeric antigen to mix and block; preferably, the pH of the solution for preparing the colloidal gold recombinant protein is 6.2; preferably, the concentration of the chimeric antigen is not less than 2 μg / mL; preferably, the blocking is performed using a 10% BSA solution.

[0032] In one embodiment, the concentration of the colloidal gold marker, the recombinant protein A or the antibody capable of binding to the chimeric antigen is 4 mg / mL.

[0033] In one embodiment, the marking volume of the test area and the quality control area is 1 μl / cm.

[0034] Beneficial Effects

[0035] The present invention provides a novel chimeric antigen of Toxoplasma gondii. When used as a diagnostic antigen for toxoplasmosis, this chimeric antigen can significantly improve the specificity and sensitivity of detection and is suitable for application in different scenarios such as on-site diagnosis and laboratory testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 . Schematic diagram of the design of multi-epitope chimeric antigens.

[0037] Figure 2 . Expression, purification and identification of multi-epitope chimeric antigens. A is the expression trend of recombinant multi-epitope chimeric antigens (rMEP), B is the purification result of rMEP protein, C is the Western blotting identification result of rMEP, and D is the rMEP reactogenicity identification result.

[0038] Figure 3 . Preparation and identification of polyclonal antibodies against recombinant multi-epitope chimeric antigens (rMEP). A is the identification of antibody reactivity, and B is the result of the determination of antibody titer.

[0039] Figure 4 .Test strip specificity test results.

[0040] Figure 5 .Dilution multiple sensitivity test results.

[0041] Figure 6 .Test strips show the sensitivity of the number of days of infection.

[0042] Figure 7 .Stability test results of the test strips. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0044] Example 1. Design of multi-epitope chimeric antigen protein

[0045] Six antigenic epitopes from Toxoplasma surface antigen 1 (SAG1), surface antigen 3 (SAG3), bradyzoite antigen 1 (BAG1), rhoptry protein 2 (ROP2), and dense granule protein 54 (GRA54) were selected and linked together through a linker to form a multiepitope fusion peptide (MEP). The sequences of each antigenic epitope and multiepitope chimeric antigen are shown in Table 1. The schematic diagram of the multiepitope chimeric antigen is shown in Figure 1 .

[0046] Table 1 Diagnostic antigen epitope sequences

[0047]

[0048]

[0049] The designed multi-epitope chimeric antigen was subjected to bioinformatics analysis, and the results showed that the multi-epitope chimeric antigen protein had a half-life of more than 10 hours in Escherichia coli and was a stable protein. It was antigenic, had no signal peptide and topological structure, and most amino acids existed in a random coil conformation, so it was speculated that the corresponding epitopes of the chimeric antigen protein were also exposed on the surface of the protein. The bioinformatics analysis results of the multi-epitope chimeric antigen protein are shown in Table 2, and the analysis results showed that the protein could be used as a diagnostic antigen for subsequent experiments.

[0050] Table 2 Bioinformatics analysis of multi-epitope chimeric antigen proteins

[0051]

[0052]

[0053] Example 2. Expression and purification of multi-epitope chimeric antigen protein

[0054] The pET-32a(+) plasmid was connected to the multi-epitope chimeric antigen protein sequence to obtain the recombinant plasmid pET-32a(+)-MEP. The recombinant plasmid pET-30a(+)-MEP was transferred into Rosetta(DE3)pLysS competent cells. After culturing on LB solid medium containing ampicillin for 12 h, the complete monoclonal colonies were picked and added to LB medium containing 20 μg / mL ampicillin. The cells were expressed on a shaker at 37°C and 190 rpm until the OD value was about 0.5. Subsequently, 0.2 mM IPTG was added and the cells were induced to express at 37°C and 200 rpm. The bacterial liquid samples at 0 h, 2 h, 4 h, 6 h, and 8 h were prepared and analyzed by SDS-PAGE. The results are shown in the figure. Figure 2 As shown in A. Figure 2 In A, M is a marker, lanes 1-5 are bacterial suspensions at 0h, 2h, 4h, 6h, and 8h, respectively, and lanes 6-7 are supernatant proteins and precipitated proteins after bacterial ultrasonic lysis, respectively. Figure 2 As shown in A, the expression level of MEP protein reached the highest level after 8 hours of induction, and MEP protein was expressed in both the supernatant and the precipitate. The protein in the supernatant was taken for subsequent experiments.

[0055] The bacterial precipitate was collected by centrifugation and ultrasonically disrupted. The supernatant was centrifuged at 4°C and 10,000 g for 10 min. The supernatant was aspirated and filtered through a 0.22 μm water filter. The supernatant was slowly added to the protein purification column. The sample was loaded 5 times. The protein was eluted with different concentrations of imidazole eluents and the eluents were collected. 20 μL and 5 μL of the eluent were taken from each tube. Prepare the sample with 5×SDS, and perform SDS-PAGE gel electrophoresis at 120V constant voltage for 50min. After the electrophoresis, remove the gel block, stain it in Coomassie Brilliant Blue staining solution for 30min, and then rinse the protein gel with decolorizing solution. Change the decolorizing solution every 5min, and repeat until the gel block background is transparent. According to the SDS-PAGE results, select several tubes of eluent with few impurities and dense target bands for protein concentration determination. Transfer the selected eluent to a dialysis bag and dialyze it in 10mMPB solution for 48h, changing the solution every 12h, and changing the solution 3 times. Centrifuge after dialysis, aspirate the supernatant, measure the concentration again, and store the protein solution at -80℃. The protein purification results are shown in the figure. Figure 2 As shown in B, Figure 2In B, M is a marker, lane 1 is the sample solution before sample purification, lane 2 is the residual solution after sample purification, lanes 3-4 are the sample solutions eluted with 10mM imidazole eluent, lanes 5-6 are the sample solutions eluted with 20mM imidazole eluent, lanes 7-8 are the sample solutions eluted with 40mM imidazole eluent, lanes 9-10 are the sample solutions eluted with 60mM imidazole eluent, and lanes 11-13 are the sample solutions eluted with 250mM imidazole eluent. It can be seen that in addition to the target band, there are also miscellaneous bands in lane 1. Lane 2 does not contain the target band, indicating that the target protein MEP is in the protein purification column. Lanes 3-4 still have many miscellaneous bands. The bands in lanes 5-10 are not obvious. Only in lanes 11-13, a single band appeared, indicating that in the sample solution eluted with 250 mM imidazole eluent, a large amount of target protein was eluted, with a size of about 31 kDa and a high purity.

[0056] Example 3: Identification of recombinant protein rMEP

[0057] In this example, the recombinant protein rMEP obtained in Example 2 was identified by Western blotting experiment, and the experimental steps included the following parts:

[0058] (1) Sample preparation: Dilute the purified rMEP protein to 0.1 mg / mL, take 16 μL of the diluted protein and mix it with 4 μL of 5× SDS, boil it in boiling water for 10 min, and then quickly transfer it to ice;

[0059] (2) Sample loading: Add 10 μL of sample to each well of the protein gel and perform electrophoresis at 120 V constant voltage for 50 min;

[0060] (3) Electrotransfer: Cut the PVDF membrane of appropriate size and electrotransfer at 0.22A constant current for 45 min;

[0061] (4) Blocking: Remove the membrane and block it in 5% skim milk at room temperature for 2 h;

[0062] (5) Washing membrane: rinse with 1× PBST three times, 5 min each time;

[0063] (6) Incubation with primary antibody: Incubate in 1:1000 diluted anti-His mouse IgG at 4°C for 12 h. After incubation, wash the membrane seven times, 5 min each time.

[0064] (7) Incubation with secondary antibody: Incubate in 1:5000 diluted rabbit anti-mouse IgG-HRP at room temperature for 1 h, wash the membrane 7 times, 5 min each time;

[0065] (8) Chemiluminescent liquid reaction and signal collection using Bio-Rad instrument.

[0066] The experimental results are as follows Figure 2 As shown in C, Figure 2 In C, M is a marker, and the rMEP band is developed to obtain a target band of 31 kDa, indicating that the recombinant protein with a His tag after recombinant expression and purification in Example 2 is the target protein rMEP.

[0067] Example 4: Identification of the reactogenicity of the recombinant protein rMEP

[0068] In this example, the reactivity of the recombinant protein rMEP obtained in Example 2 was identified by Western blotting experiment. The experimental steps included the following parts:

[0069] (1) Sample preparation: Dilute the purified protein rMEP to 0.1 mg / mL, take 16 μL of the diluted protein and mix it with 4 μL of 5× SDS, boil it in boiling water for 10 min, and then quickly transfer it to ice;

[0070] (2) Sample loading: Add 10 μL of sample to each well of the protein gel and perform electrophoresis at 120 V constant voltage for 50 min;

[0071] (3) Electrotransfer: Cut the PVDF membrane of appropriate size and electrotransfer at 0.22A constant current for 45 min;

[0072] (4) Blocking: Remove the membrane and block it in 5% skim milk at room temperature for 2 h;

[0073] (5) Washing membrane: rinse with 1× PBST three times, 5 min each time;

[0074] (6) Incubation with primary antibody: Incubate in 1:3000 diluted mouse Toxoplasma gondii positive serum and mouse Toxoplasma gondii negative serum at 4°C for 12 h. After the incubation, wash the membrane 7 times, 5 min each time.

[0075] (7) Incubation with secondary antibody: Incubate in 1:5000 diluted rabbit anti-mouse IgG-HRP at room temperature for 1 h, wash the membrane 7 times, 5 min each time;

[0076] (8) Chemiluminescent liquid reaction and signal collection using Bio-Rad instrument.

[0077] The experimental results are as follows Figure 2 As shown in D, Figure 2 In D, the primary antibody of the Positive group is mouse Toxoplasma positive serum, and the primary antibody of the Negative group is mouse Toxoplasma negative serum. After color development, a band was detected at the target size in the Positive group, while no band was detected in the Negative group, indicating that the mouse Toxoplasma positive serum contains antibodies that can recognize the above rMEP, while the negative serum does not have antibodies that can recognize the above rMEP. That is, the above rMEP can recognize and bind to the antibodies in the Toxoplasma positive serum, and the above rMEP has antigenicity.

[0078] Example 5. Preparation and purification of polyclonal antibodies

[0079] (1) 2.5 kg male New Zealand white rabbits were selected and subcutaneously injected with emulsified recombinant multi-epitope chimeric antigen (rMEP) for polyclonal antibody preparation. The first immunization was performed with an equal volume of rMEP emulsified with Freund's complete adjuvant at a dose of 500 μg / rabbit; two weeks later, the second immunization was performed with an equal volume of rMEP emulsified with Freund's incomplete adjuvant at a dose of 250 μg / rabbit; ten days later, the third immunization was performed with the same dose as the second immunization; blood was collected one week later, and serum was obtained to obtain the polyclonal antibody before purification.

[0080] (2) After thawing the serum on ice, measure the volume and add it to a beaker. Add three times the volume of the serum into the beaker with acetate buffer (0.06 M, pH = 4.6);

[0081] (3) Add octanoic acid dropwise under magnetic stirring (500 mL serum plus 17 mL octanoic acid) and stir for 30 min;

[0082] (4) Collect the supernatant by centrifugation, put it into a dialysis bag and dialyze it in 0.01 M PB (pH = 7.4) solution, change the water three times, and store at 4°C overnight;

[0083] (5) Remove the dialysis bag, measure the total volume of serum and PB, add an equal volume of ammonium sulfate dropwise, centrifuge, discard the supernatant, resuspend the precipitate with 0.01 M PB solution, dialyze in 0.01 M PB solution, and centrifuge. Collect the supernatant to obtain the purified polyclonal antibody, which is stored in a -80°C refrigerator.

[0084] Example 6. Determination of polyclonal antibody reactivity and titer

[0085] Western blotting was used to verify the reactivity of the polyclonal antibody. The experimental steps were the same as in Example 3. The sample was prepared using recombinant multi-epitope chimeric antigen (rMEP), the purified polyclonal antibody diluted 1:3000 was used as the primary antibody, and the goat anti-rabbit IgG-HRP diluted 1:5000 was used as the secondary antibody. The experimental results after color development are as follows: Figure 3 As shown in A, M stands for Maker. A band appeared at 31 kDa in the rMEP lane, indicating that the prepared polyclonal antibody can bind to rMEP and has good reactivity.

[0086] The ELISA method was used to measure the titer of polyclonal antibodies before and after purification. The ELISA detection steps are as follows:

[0087] (1) Dilute rMEP to 5 μg / mL with coating solution, add the dilution solution to a 96-well plate at 100 μL / well, place the 96-well plate in a wet box, and incubate at 4°C for 12 h;

[0088] (2) Discard the diluent and add PBST solution to wash the 96-well plate, 230 μL / well, wash 5 times, 3 min / time, and pat the 96-well plate dry after washing;

[0089] (3) Add 5% skim milk for blocking, 100 μL / well, and block at 37°C for 2 h;

[0090] (4) diluting the polyclonal antibody before and after purification in multiple ratios with 5% skim milk;

[0091] (5) Discard the blocking solution and add PBST solution to wash the 96-well plate, 230 μL / well, wash 5 times, 3 min / time, and pat the 96-well plate dry after washing;

[0092] (6) Add diluted pre-purification or post-purification polyclonal antibody as primary antibody, 100 μL / well, and incubate at 37°C for 1 h;

[0093] (7) Discard the primary antibody and add PBST solution to wash the 96-well plate, 230 μL / well, wash 5 times, 3 min / time, and pat the 96-well plate dry after washing;

[0094] (8) Add 1:5000 diluted goat anti-rabbit IgG-HRP as secondary antibody, 100 μL / well, and incubate at 37°C for 45 min;

[0095] (9) Discard the secondary antibody and add PBST solution to wash the 96-well plate, 230 μL / well, wash 5 times, 3 min / time, and pat the 96-well plate dry after washing;

[0096] (10) Add 100 μL / well of colorimetric solution and react at 37°C in the dark for 15 min.

[0097] (11) Add 2 M sulfuric acid solution to terminate the reaction, 100 μL / well;

[0098] (12) Measure the OD of each well using an ELISA reader 450 , P / N>2.1 (the OD value of the experimental group is 2.1 times greater than the OD value of the negative control well) was judged as positive.

[0099] The experimental results are as follows Figure 3 As shown in B, the titer of the polyclonal antibody was lost after purification, but the highest titer of the polyclonal antibody before and after purification could reach 1:4096000, indicating that the purified polyclonal antibody had a strong binding force with rMEP and could be used as the antibody for the quality control line of the colloidal gold immunochromatography test strip.

[0100] Example 7. Preparation of colloidal gold immunochromatographic test strips

[0101] The present application uses the trisodium citrate reduction method to prepare colloidal gold particles of appropriate size, and uses the recombinant chimeric protein rMEP as the gold-labeled antigen. The preparation steps are as follows:

[0102] 1. Preparation of colloidal gold particles: Add 500 mL of distilled water to a clean conical flask, place it on a heatable magnetic stirrer, and boil it; weigh 0.118 g of sodium citrate and dissolve it in 1 mL of boiling water; add 8.2 mL of 1% chloroauric acid to the conical flask; quickly add the sodium citrate solution to the conical flask, react for 10 minutes, and observe the color change; the prepared colloidal gold solution is stored at 4°C for later use.

[0103] 2. Dilute recombinant protein A (purchased from Sangon Biotechnology, catalog number C610039) to 4 mg / mL for marking the test line (T line). Dilute rMEP polyclonal antibody to 4 mg / mL for quality control line (C line). Paste NC membrane (nitrocellulose membrane) on the base plate, and then use colloidal gold spotting system for marking. The marking volume is 1 μl / cm. After marking, place the base plate at 37°C overnight for drying, seal it in a bag, and build in desiccant.

[0104] 3. Determination of the optimal pH for colloidal gold labeling of recombinant protein

[0105] Prepare 8 centrifuge tubes, add 1mL colloidal gold solution to each tube, add 0, 1, 2, 3, 4, 5, 6, 7μL 0.2MK2CO3 respectively, mix well; add 10μg recombinant protein to each tube, mix well; add 100μL 10% NaCl solution to each tube, mix well, and observe the color change of the colloidal gold solution. By observing the color change of the colloidal gold solution, it can be seen that when 2μL K2CO3 is added, the color of the solution no longer changes. When actually marking, take the last tube of the color-stable tube, that is, the 4th tube, and use pH test paper to measure the pH of the solution to be 6.2, which shows that the optimal pH for labeling rMEP protein is 6.2.

[0106] 4. Determination of the optimal amount of labeled protein

[0107] Prepare 8 centrifuge tubes, add 1mL colloidal gold solution to each tube and adjust to the optimal pH 6.2 with 0.2M K2CO3, mix well; add 0, 2, 4, 6, 8, 10, 12, 14μg recombinant protein to each tube, mix well; add 100μL 10% NaCl solution to each tube, mix well, and observe the color change of the colloidal gold solution. When 2μg / mL rMEP protein is added, the colloidal gold solution no longer changes color; the minimum labeling amount of protein is 2μg / mL. Generally, in practical applications, at least 10% to 20% more protein is added, so the optimal labeling amount of rMEP protein is 4μg / mL.

[0108] 5. Colloidal gold labeled protein: Take a 15mL centrifuge tube, add 10mL colloidal gold solution, adjust to the optimal labeling pH 6.2 with 0.2M K2CO3, add the optimal amount of recombinant multi-epitope chimeric antigen rMEP protein (4μg / mL), mix well, and let stand for 30 minutes; add 1mL 10% BSA solution, mix well, and block at 4°C for 12 hours; after blocking, centrifuge at 4°C and 10000g for 30 minutes, discard the supernatant, and resuspend the bottom floccules with 500μL gold label diluent. Store the gold-labeled protein at 4°C for later use.

[0109] 6. Sample pad pretreatment: Soak the sample pad in a phosphate system sample pad treatment solution with a pH of 7.5 for 30 minutes, and then dry it at 37°C overnight for use.

[0110] 7. Finally, the reaction membrane, gold label pad, sample pad and water absorbent pad are sequentially bonded to the PVC base plate and cut into strips with a width of 4 mm to make colloidal gold immunochromatographic test strips for detecting Toxoplasma gondii infection.

[0111] Example 8: Testing of colloidal gold test strip performance

[0112] 1. Specificity test: dilute rat Toxoplasma negative and positive serum, dog Toxocara positive serum, sheep Haemonchus contortus positive serum, gerbil Echinococcus positive serum, sheep Fasciola positive serum with PB 20 times, take 40μL and drop it on the colloidal gold test paper sample pad, and determine the result within 10 minutes. The experimental results are as follows Figure 4 As shown, in Figure 4 Among them, 1 is rat Toxoplasma negative serum, 2 is rat Toxoplasma positive serum, 3 is dog Toxocara positive serum, 4 is sheep Haemonchus contortus positive serum, 5 is mouse Echinococcus positive serum, 6 is sheep Fasciola positive serum. It can be seen that only Toxoplasma positive serum can turn the T line of the test strip red, indicating that the test strip has good specificity.

[0113] 2. Test of sensitivity of serum dilution multiple: dilute the Toxoplasma positive serum with PB at the ratio of 1:40, 1:80, 1:160, 1:320, 1:640, 1:800, 1:1000, 1:1200, take 40μL of each and drop it on the colloidal gold test strip sample pad, and determine the result within 10 minutes. The experimental results are as follows Figure 5 As shown, in Figure 5 In the table, 1 is Toxoplasma-negative serum, 2-10 are Toxoplasma-positive serum with dilution ratios of 1:40, 1:80, 1:160, 1:320, 1:640, 1:800, 1:1000, and 1:1200, respectively. The highest dilution multiple at which a positive reaction was observed was 1:800.

[0114] 3. Test of sensitivity of infection days: Three 4-6 week old F344 female rats were artificially infected and intraperitoneally injected with 10 5 The ME49 Toxoplasma gondii tachyzoites were collected from one of the rats on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 after infection. After incubation at 37°C for two hours, the blood was centrifuged at 4000g for 10 minutes, and the serum was pipetted into a new EP tube and stored at -80°C. After diluting 40 times with PB, 40 μL was added to the sample pad of the test strip and the color change of the T line was observed. The experimental results are shown in Figure 2. Figure 6 As shown, it can be seen that the T line of the test strip begins to turn red 13 days after infection.

[0115] 4. Stability test: Store the test strips at 4°C for 30 and 60 days to test their stability. Figure 7 As shown, the test strip can still detect positive results at 4°C for 60 days, indicating good stability.

[0116] Example 9: Comparative test of multi-epitope chimeric diagnostic antigen and single-epitope diagnostic antigen colloidal gold test strips

[0117] The 6 antigen epitopes of the multi-epitope chimeric antigen protein in Example 1 were expressed and purified by prokaryotic expression, and colloidal gold test strips were made in the same way, and the sensitivity of serum dilution multiples was tested. The Toxoplasma gondii positive serum tested in Example 8 was diluted with PB at a ratio of 1:40, 1:80, 1:160, 1:320, 1:640, 1:800, 1:1000, and 1:1200, and 40 μL was added to the colloidal gold test strip sample pad, and the result was determined within 10 minutes. The experimental results are shown in Table 3. The sensitivity of the single epitope diagnostic antigen is relatively low, while the sensitivity of the multi-epitope chimeric diagnostic antigen is high, indicating that the sensitivity of the single epitope diagnostic antigen is not as good as that of the multi-epitope chimeric diagnostic antigen.

[0118] Table 3 Sensitivity results of colloidal gold test strips with single epitope diagnostic antigens and multi-epitope chimeric diagnostic antigens

[0119]

[0120] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A Toxoplasma gondii chimeric antigen, characterized in that: The chimeric antigen includes antigenic epitopes of surface antigen 1, surface antigen 3, bradyzoite antigen 1, rhoptry protein 2 and dense granule protein 54.

2. The chimeric antigen according to claim 1, characterized in that The sequence of the chimeric antigen is shown as SEQ ID No.

8.

3. Use of the chimeric antigen according to any one of claims 1 to 2 in the preparation of a toxoplasmosis detection kit.

4. A colloidal gold immunochromatographic test strip for detecting toxoplasmosis, characterized in that: The test strip uses the chimeric antigen described in any one of claims 1-2 as the gold-labeled antigen.

5. The test strip according to claim 4, characterized in that: The test strip comprises a reaction membrane, a sample pad, a gold label pad, a water absorbent pad and a backing, and the gold label pad is coated with a colloidal gold label of the chimeric antigen according to any one of claims 1-2.

6. The test strip according to claim 5, characterized in that: The reaction membrane is coated with a quality control area of ​​an antibody that can bind to the chimeric antigen according to any one of claims 1-2.

7. The test strip according to claim 5, characterized in that: The reaction membrane is provided with a test area coated with recombinant protein A.

8. The test strip according to claim 5, characterized in that: The reaction membrane is a NC membrane (nitrocellulose membrane).

9. The test strip according to any one of claims 4 to 8, characterized in that: The test strip also includes a loading card.

10. A method for preparing a colloidal gold immunochromatographic test strip for detecting toxoplasmosis, characterized in that: The method comprises the steps of: 1) Expression and purification of the chimeric antigen according to any one of claims 1-2; 2) preparing a gold label pad, wherein the gold label pad is coated with a colloidal gold label of the chimeric antigen according to any one of claims 1 to 2; 3) preparing a reaction membrane comprising a test area coated with recombinant protein A and a quality control area coated with an antibody capable of binding to the chimeric antigen according to any one of claims 1 to 2; 4) Assembling the prepared gold label pad, reaction membrane, sample pad, water absorbent pad and backing into the test strip.

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